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50:T3c22,Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation Dongsheng Luo & Efstathios S. Giotis School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK 1. Abstract Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3]. Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii, and Plecotus auritus, representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling (Fig.1 ). We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing. Fig.1 Integrated workflow 2. Introduction Background and rationale Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies. The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance. This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project (https://bat1k.com) and the BatProtect consortium (https://cordis.europa.eu/project/id/101118919). The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses. Hypotheses H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species. Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species. H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species. Primary outcome: differentially expressed genes shared across multiple species following stimulation. H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes. Primary outcome: species-specific or differentially regulated genes and pathways following stimulation. H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation. This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility. 3. Methods Participants/Sample This study does not involve human participants or new animal experimentation. The experimental samples consist of established bat cell cultures from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii and Plecotus auritus. Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design. All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards. Experimental Design and Sequencing Metrics The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing. For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2: Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples Sequencing Specifications: • Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system). • Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp). • Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample. • Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing. Materials and Procedures Work Package 1: RNA quality control and validation of innate immune activation Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction. Work Package 2: Comparative RNA sequencing of bat innate immune responses All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek® Flow® software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant. The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow: 1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment. 2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium. 3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow. Work Package 3: Discovery and prioritisation of candidate antiviral ISGs Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into: • Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages. • Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis). • Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation. Package 4: Data integration and dissemination The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes. Ethics and Data Management The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted. 4. Pilot Data (Ongoing Preliminary Experiment) The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway. The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions. This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study. 5. Work Packages and Timeline Project duration: 9 months Work Package / Activity M1 M2 M3 M4 M5 M6 M7 M8 M9 WP1: RNA QC and qPCR validation ■ ■ WP2: RNA-seq & Partek Flow bioinformatics ■ ■ ■ ■ WP3: ISG discovery, Bat1K analysis ■ ■ ■ WP4: Integration and dissemination ■ ■ Key milestones Milestone Description Target MS1 RNA QC and innate immune response validation completed End of Month 2 MS2 RNA sequencing completed or underway End of Month 3 MS3 Species-specific differential expression analysis completed End of Month 5 MS4 Candidate ISGs prioritised and key findings integrated End of Month 7 MS5 Dataset and analysis outputs prepared for dissemination End of Month 9 6. Budget Proposed budget: $10,000 Budget Item Details & Justification Estimated Cost (USD) RNA Quality Control & Sample Prep Bioanalyzer chips, NanoDrop consumables, quantification kits $400 RT-qPCR Validation Reagents cDNA synthesis kits, SYBR Green master mixes, validated bat primers $1,200 RNA-seq Library Preparation Stranded mRNA library preparation kits for 24 samples $2,000 RNA Sequencing (NovaSeq PE150) Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files) $5,000 Targeted Validation & Functional Reagents Expanded independent RT-qPCR validation assays and molecular reagents $900 General Laboratory Consumables Pipette tips, nuclease-free plasticware, cold-chain reagents $500 Bioinformatics & Partek Flow® Licences Covered by existing lab infrastructure & institutional compute $0 Total $10,000 Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek® Flow® software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding). 7. Expected Impact This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design. 8. References 1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192. 2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214. 3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113. 4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0. 5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23.51:T3e5e,Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation Dongsheng Luo & Efstathios S. Giotis School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK 1. Abstract Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3]. Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii, and Plecotus auritus, representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling (Fig.1 ). We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing. Fig.1 Integrated workflow 2. Introduction Background and rationale Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies. The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance. This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project (https://bat1k.com) and the BatProtect consortium (https://cordis.europa.eu/project/id/101118919). The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses. Hypotheses H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species. Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species. H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species. Primary outcome: differentially expressed genes shared across multiple species following stimulation. H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes. Primary outcome: species-specific or differentially regulated genes and pathways following stimulation. H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation. This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility. 3. Methods Participants/Sample This study does not involve human participants or new animal experimentation. The experimental samples consist of established bat cell cultures from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii and Plecotus auritus. Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design. All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards. Experimental Design and Sequencing Metrics The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing. For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2: Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples Sequencing Specifications: • Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system). • Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp). • Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample. • Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing. Materials and Procedures Work Package 1: RNA quality control and validation of innate immune activation Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction. Work Package 2: Comparative RNA sequencing of bat innate immune responses All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek® Flow® software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant. The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow: 1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment. 2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium. 3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow. Work Package 3: Discovery and prioritisation of candidate antiviral ISGs Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into: • Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages. • Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis). • Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation. Package 4: Data integration and dissemination The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes. Ethics and Data Management The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted. 4. Pilot Data (Ongoing Preliminary Experiment) The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway. The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions. This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study. 5. Work Packages and Timeline Project duration: 9 months Work Package / Activity M1 M2 M3 M4 M5 M6 M7 M8 M9 WP1: RNA QC and qPCR validation ■ ■ WP2: RNA-seq & Partek Flow bioinformatics ■ ■ ■ ■ WP3: ISG discovery, Bat1K analysis ■ ■ ■ WP4: Integration and dissemination ■ ■ Key milestones Milestone Description Target MS1 RNA QC and innate immune response validation completed End of Month 2 MS2 RNA sequencing completed or underway End of Month 3 MS3 Species-specific differential expression analysis completed End of Month 5 MS4 Candidate ISGs prioritised and key findings integrated End of Month 7 MS5 Dataset and analysis outputs prepared for dissemination End of Month 9 6. Budget Proposed budget: $10,000 Budget Item Details & Justification Estimated Cost (USD) RNA Quality Control & Sample Prep Bioanalyzer chips, NanoDrop consumables, quantification kits $400 RT-qPCR Validation Reagents cDNA synthesis kits, SYBR Green master mixes, validated bat primers $1,200 RNA-seq Library Preparation Stranded mRNA library preparation kits for 24 samples $2,000 RNA Sequencing (NovaSeq PE150) Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files) $5,000 Targeted Validation & Functional Reagents Expanded independent RT-qPCR validation assays and molecular reagents $900 General Laboratory Consumables Pipette tips, nuclease-free plasticware, cold-chain reagents $500 Bioinformatics & Partek Flow® Licences Covered by existing lab infrastructure & institutional compute $0 Total $10,000 Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek® Flow® software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding). 7. Expected Impact This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design. 8. References 1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192. 2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214. 3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113. 4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0. 5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23. 52:T1486a,{"type":"doc","content":[{"type":"heading","attrs":{"id":"3114926e-7a4f-41c6-8ba7-5b8ee1ef0335","data-toc-id":"3114926e-7a4f-41c6-8ba7-5b8ee1ef0335","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"18pt","fontFamily":"","color":"black"}},{"type":"bold"}],"text":"Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation"}]},{"type":"heading","attrs":{"id":"64e5a1a5-e226-4d91-985c-73644993f9e2","data-toc-id":"64e5a1a5-e226-4d91-985c-73644993f9e2","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Dongsheng Luo & Efstathios S. Giotis"}]},{"type":"paragraph","attrs":{"id":"3b5ce86e-a4e2-47ba-8b02-949a86c58e8a","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":""}}],"text":"School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK"}]},{"type":"heading","attrs":{"id":"1332fbc7-5d7d-43d7-92b8-83cfa8cd8350","data-toc-id":"1332fbc7-5d7d-43d7-92b8-83cfa8cd8350","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"1. Abstract"}]},{"type":"paragraph","attrs":{"id":"26e2087b-3865-4a9a-b5f5-0b637ec302d3","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3]."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Myotis myotis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Rhinolophus ferrumequinum"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Myotis bechsteinii"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Plecotus auritus"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling ("},{"type":"text","marks":[{"type":"bold"}],"text":"Fig.1"},{"type":"text","text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":")."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing."}]},{"type":"imageBlock","attrs":{"src":"https://storage.prod.researchhub.com/uploads/notes/users/207668/7f21793a-2fd8-4604-8f5d-4b23da25077d/image.png","width":"100%","align":"left"}},{"type":"paragraph","attrs":{"id":"1d751b3f-b77c-4117-aa22-3b41e5c16877","class":null,"textAlign":"center"},"content":[{"type":"text","marks":[{"type":"bold"}],"text":"Fig.1"},{"type":"text","text":" Integrated workflow"}]},{"type":"heading","attrs":{"id":"94b7fb54-9fea-4faf-9234-e7ad13fd8f1a","data-toc-id":"94b7fb54-9fea-4faf-9234-e7ad13fd8f1a","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"2. Introduction"}]},{"type":"heading","attrs":{"id":"228101a2-90c2-4b2a-ab7e-a92b3772625b","data-toc-id":"228101a2-90c2-4b2a-ab7e-a92b3772625b","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Background and rationale"}]},{"type":"paragraph","attrs":{"id":"9ffad870-a4ce-457f-9a23-7b9fe9b82fc6","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance."}]},{"type":"paragraph","attrs":{"id":"48112859-fa4c-49b8-b3ab-65f1e73f69c0","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project ("},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://bat1k.com","target":"_blank","rel":"noopener noreferrer nofollow","class":"link","title":null}},{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":""}},{"type":"underline"}],"text":"https://bat1k.com"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":") and the BatProtect consortium ("},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://cordis.europa.eu/project/id/101118919","target":"_blank","rel":"noopener noreferrer nofollow","class":"link","title":null}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"blue"}},{"type":"underline"}],"text":"https://cordis.europa.eu/project/id/101118919"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":")."}]},{"type":"paragraph","attrs":{"id":"21f3b9f4-f371-4ccd-9fdd-665ffa70bfe8","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses."}]},{"type":"heading","attrs":{"id":"61c26901-7322-4452-8ab9-28b247bd813a","data-toc-id":"61c26901-7322-4452-8ab9-28b247bd813a","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Hypotheses"}]},{"type":"heading","attrs":{"id":"8d741253-4483-43fb-92c8-192da4bfac63","data-toc-id":"8d741253-4483-43fb-92c8-192da4bfac63","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species."}]},{"type":"paragraph","attrs":{"id":"1a393cc3-4621-4459-9669-b3c7db7c78ec","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species."}]},{"type":"heading","attrs":{"id":"ebd0b3ca-42fa-4020-bb08-24aa041a784b","data-toc-id":"ebd0b3ca-42fa-4020-bb08-24aa041a784b","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species."}]},{"type":"paragraph","attrs":{"id":"1498c99c-6847-4270-9448-fd133c45936f","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Primary outcome: differentially expressed genes shared across multiple species following stimulation."}]},{"type":"heading","attrs":{"id":"094888d1-a741-4ff8-ace3-7d45a96277ee","data-toc-id":"094888d1-a741-4ff8-ace3-7d45a96277ee","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes."}]},{"type":"paragraph","attrs":{"id":"fae98a17-c0a6-4173-bc8a-2d629c2aa860","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Primary outcome: species-specific or differentially regulated genes and pathways following stimulation."}]},{"type":"heading","attrs":{"id":"b1bd33a5-4060-43dc-9fe0-f49ca17159a6","data-toc-id":"b1bd33a5-4060-43dc-9fe0-f49ca17159a6","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation."}]},{"type":"paragraph","attrs":{"id":"3b4627cc-dc76-453c-a98b-26e324c316de","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility."}]},{"type":"heading","attrs":{"id":"cd09551c-c2d7-4ab5-8fe7-a433828a5c08","data-toc-id":"cd09551c-c2d7-4ab5-8fe7-a433828a5c08","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"3. Methods"}]},{"type":"heading","attrs":{"id":"93dd2c13-0440-4428-bd5a-0ddde6a91a48","data-toc-id":"93dd2c13-0440-4428-bd5a-0ddde6a91a48","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Participants/Sample"}]},{"type":"paragraph","attrs":{"id":"afcd77eb-1fbb-4b5f-b3c1-98ee02f6d09d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This study does not involve human participants or new animal experimentation."}]},{"type":"paragraph","attrs":{"id":"af9406f0-ccc8-4ed8-ba04-83369dd21815","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The experimental samples consist of established bat cell cultures from four European bat species: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Myotis myotis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Rhinolophus ferrumequinum, Myotis bechsteinii "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"and"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":" Plecotus auritus"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":". Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design."}]},{"type":"paragraph","attrs":{"id":"71359f18-0fdc-4260-a268-20f00cad6c03","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards."}]},{"type":"heading","attrs":{"id":"2d63fe3d-9684-420e-b7df-1b9e1e19701b","data-toc-id":"2d63fe3d-9684-420e-b7df-1b9e1e19701b","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Experimental Design and Sequencing Metrics"}]},{"type":"paragraph","attrs":{"id":"1f97f8cc-e570-4e45-a4d6-2c28cd9fd562","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing."}]},{"type":"paragraph","attrs":{"id":"5a9ae4f0-4437-45d2-93e3-f62849e6e188","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2:"}]},{"type":"paragraph","attrs":{"id":"f92f9332-71dc-4ba8-b45d-dc00b5113206","class":"MsoNormal","textAlign":"center"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"bold"}],"text":"Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples"}]},{"type":"paragraph","attrs":{"id":"1b4fd621-187e-4cad-bc06-f2de626e8948","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Sequencing Specifications:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system)."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp)."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing."}]},{"type":"paragraph","attrs":{"id":"78aafbb2-b390-41f4-98d1-b8da4859bc0d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"11pt","fontFamily":"","color":"black"}}],"text":" "}]},{"type":"heading","attrs":{"id":"7d729a67-c1a3-47ac-9b34-38e65953e2a4","data-toc-id":"7d729a67-c1a3-47ac-9b34-38e65953e2a4","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Materials and Procedures"}]},{"type":"heading","attrs":{"id":"0be9bdf4-3993-4c98-a4a9-36c9350fd866","data-toc-id":"0be9bdf4-3993-4c98-a4a9-36c9350fd866","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Work Package 1: RNA quality control and validation of innate immune activation"}]},{"type":"paragraph","attrs":{"id":"77195db0-31ad-4569-b498-eca55a5bde8c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction."}]},{"type":"heading","attrs":{"id":"df923cef-33a0-457a-8b22-5accf066d9e9","data-toc-id":"df923cef-33a0-457a-8b22-5accf066d9e9","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Work Package 2: Comparative RNA sequencing of bat innate immune responses"}]},{"type":"paragraph","attrs":{"id":"17760ed7-effb-489c-99a0-581e2bf79e6f","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" Flow"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant."}]},{"type":"paragraph","attrs":{"id":"36d48ef8-4918-4baf-a6de-2552c90e6915","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow:"}]},{"type":"paragraph","attrs":{"id":"41d6e956-5ed9-4da7-a6f6-846a5b71b9cd","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment."}]},{"type":"paragraph","attrs":{"id":"83a822a7-4263-46dd-8fd2-a11c852ff703","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium."}]},{"type":"paragraph","attrs":{"id":"bd9a8815-4ee8-4409-ac8c-91e4ff61d743","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow."}]},{"type":"heading","attrs":{"id":"1bae9cbb-05d2-4bfe-af59-143eb5a1a5f6","data-toc-id":"1bae9cbb-05d2-4bfe-af59-143eb5a1a5f6","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Work Package 3: Discovery and prioritisation of candidate antiviral ISGs"}]},{"type":"paragraph","attrs":{"id":"c215bb45-9289-4dc7-8821-ab363f9368e4","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into:"}]},{"type":"paragraph","attrs":{"id":"760432e6-facd-4474-886c-a6cab33fada7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages."}]},{"type":"paragraph","attrs":{"id":"b81b8a79-4372-4df4-abc9-936ea3a1211e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis)."}]},{"type":"paragraph","attrs":{"id":"ab7d97dc-4856-47c8-98fd-29f25403d725","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation."}]},{"type":"heading","attrs":{"id":"670a48ca-3530-4998-96c5-2ce4a6436a5c","data-toc-id":"670a48ca-3530-4998-96c5-2ce4a6436a5c","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Package 4: Data integration and dissemination"}]},{"type":"paragraph","attrs":{"id":"1645997c-2e3d-48f2-9796-cd1aa9a25f75","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes."}]},{"type":"paragraph","attrs":{"id":"e1a00751-d424-417f-98e2-2876cfb62cf2","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":" "}]},{"type":"heading","attrs":{"id":"83b69c0c-3ca4-42de-b40f-024b21e847f4","data-toc-id":"83b69c0c-3ca4-42de-b40f-024b21e847f4","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Ethics and Data Management"}]},{"type":"paragraph","attrs":{"id":"43348c2c-60eb-4153-8f6d-cbab2e314ccb","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted."}]},{"type":"heading","attrs":{"id":"5e03900a-e596-4017-b699-371413e262fc","data-toc-id":"5e03900a-e596-4017-b699-371413e262fc","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"4. Pilot Data (Ongoing Preliminary Experiment)"}]},{"type":"paragraph","attrs":{"id":"a87f12ed-46e0-4c5b-907d-547c4e958981","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study."}]},{"type":"heading","attrs":{"id":"e1bad060-0b6a-46e5-923b-b0ce175825dd","data-toc-id":"e1bad060-0b6a-46e5-923b-b0ce175825dd","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"5. Work Packages and Timeline"}]},{"type":"paragraph","attrs":{"id":"b5cc5d33-8d04-43d2-bbee-50df3dd2fdf7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Project duration: 9 months"}]},{"type":"table","attrs":{"id":"f6e9f1a8-fa9d-4918-8281-f5f6956f5054"},"content":[{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":[156],"style":"width: 195.2pt; border: 1pt solid windowtext; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 25.9pt;"},"content":[{"type":"paragraph","attrs":{"id":"c91159c7-3bfc-48a0-a2b3-84646afe56e8","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Work Package / Activity"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":[25],"style":"width: 26.1pt; border-width: 1pt 1pt 1pt medium; 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border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt;"},"content":[{"type":"paragraph","attrs":{"id":"0d788675-8294-408f-9b45-ae5e69bf6572","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Dataset and analysis outputs prepared for dissemination"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 108.25pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt;"},"content":[{"type":"paragraph","attrs":{"id":"99706d5c-a614-44a1-98dd-c75f84b71b13","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"End of Month 9"}]}]}]}]},{"type":"heading","attrs":{"id":"f83650ff-8d89-4b46-b579-b5c1f435503e","data-toc-id":"f83650ff-8d89-4b46-b579-b5c1f435503e","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"6. Budget"}]},{"type":"paragraph","attrs":{"id":"64f44e46-63dc-46f7-837e-6dbf5f93b7cf","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Proposed budget: $10,000"}]},{"type":"table","attrs":{"id":"0134fb1c-fc2c-44fb-af27-cc46a733b974"},"content":[{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border: 1pt solid windowtext; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 9.65pt;"},"content":[{"type":"paragraph","attrs":{"id":"b5b2ae91-4df2-45ad-8b7d-6cce2b68eade","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Budget Item"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-color: windowtext windowtext windowtext currentcolor; border-image: none; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 9.65pt;"},"content":[{"type":"paragraph","attrs":{"id":"88112b04-034c-4896-b988-b1a8d1483d36","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Details & Justification"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-color: windowtext windowtext windowtext currentcolor; border-image: none; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 9.65pt;"},"content":[{"type":"paragraph","attrs":{"id":"d3a0f446-ae20-4ff1-b4c8-3923ab50511b","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Estimated Cost (USD)"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"c1465ff7-37bc-4eeb-b188-b4c6998d54d2","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RNA Quality Control & Sample Prep"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"292afb34-22d2-4a31-b73b-37620758522c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Bioanalyzer chips, NanoDrop consumables, quantification kits"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"8a42681c-3d29-4c43-8f87-8d9f274186a7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$400"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"d480c40f-66ba-4462-a8b5-b2c772b80d8e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RT-qPCR Validation Reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"010bbec9-fd19-4741-9f91-7785475f6082","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"cDNA synthesis kits, SYBR Green master mixes, validated bat primers"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"e1a22ebc-3558-471e-a370-c5101d902a9c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$1,200"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"7bd0bd3e-65f6-408b-824b-219ef56641c5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RNA-seq Library Preparation"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"a495180b-623c-49f1-8162-ce4b32e6f049","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Stranded mRNA library preparation kits for 24 samples"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"328090ad-585d-443e-a616-f0a961c4d8a6","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$2,000"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 38.75pt;"},"content":[{"type":"paragraph","attrs":{"id":"1aff34b1-e286-4634-83f3-324ce04bcf0a","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RNA Sequencing (NovaSeq PE150)"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 38.75pt;"},"content":[{"type":"paragraph","attrs":{"id":"8a0828e5-b61c-4097-a651-fb58da83a700","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files)"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 38.75pt;"},"content":[{"type":"paragraph","attrs":{"id":"01dfc661-f390-4c9e-846a-c56b312372e9","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$5,000"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"add3741f-0190-41e6-a63d-ef627852cbb7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Targeted Validation & Functional Reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"28637b8c-1b51-47ca-a0d6-0a529917c968","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Expanded independent RT-qPCR validation assays and molecular reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"173e0792-d5ab-499e-91bd-801a2e66e1d1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$900 "}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.7pt;"},"content":[{"type":"paragraph","attrs":{"id":"e561a617-c808-4573-902c-65ec5dbfbb7d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"General Laboratory Consumables"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.7pt;"},"content":[{"type":"paragraph","attrs":{"id":"1dd50f80-88b3-489e-88de-4abef7d86ef5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Pipette tips, nuclease-free plasticware, cold-chain reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.7pt;"},"content":[{"type":"paragraph","attrs":{"id":"e93951c8-cfa5-40da-9e94-a437f6f26caf","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$500"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"ad6f8c74-1598-4a24-b6eb-52580facb014","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Bioinformatics & Partek Flow"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","text":" Licences"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"0a6b0c51-35d7-43ac-8e6f-1e6fa533291e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Covered by existing lab infrastructure & institutional compute"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"8cc96634-a2ac-495c-a60f-0003598090b3","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$0"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 21.25pt;"},"content":[{"type":"paragraph","attrs":{"id":"5823d16e-3e57-4b2f-bf56-3dcce2f94b10","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Total"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 21.25pt;"},"content":[{"type":"paragraph","attrs":{"id":"bb024584-8505-456d-86cc-24cba28b6696","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":" "}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 21.25pt;"},"content":[{"type":"paragraph","attrs":{"id":"01fc23a8-3f66-4514-b3d0-7ce1449f0af5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$10,000"}]}]}]}]},{"type":"paragraph","attrs":{"id":"a226e565-52f4-4781-bb01-d8eff254fbc8","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" Flow"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding)."}]},{"type":"heading","attrs":{"id":"8482e1ad-bb6e-4f05-a0ed-d5ab1fdd8644","data-toc-id":"8482e1ad-bb6e-4f05-a0ed-d5ab1fdd8644","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"7. Expected Impact"}]},{"type":"paragraph","attrs":{"id":"d1de65d2-d420-42a9-92ec-e7274ae347e5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design."}]},{"type":"heading","attrs":{"id":"8b09d553-9d07-4ba4-95e6-e68bd43b6e8b","data-toc-id":"8b09d553-9d07-4ba4-95e6-e68bd43b6e8b","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"8. References"}]},{"type":"paragraph","attrs":{"id":"e5a4cbd2-b808-4d8d-8300-f91cd1926a26","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192."}]},{"type":"paragraph","attrs":{"id":"7bf55c4c-9f14-41dd-89e7-9ced36f8b81d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214."}]},{"type":"paragraph","attrs":{"id":"13b9bd7d-9915-4823-9062-90cf325510b1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113."}]},{"type":"paragraph","attrs":{"id":"37f96245-b1ef-442d-9950-006fe5afd75a","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0."}]},{"type":"paragraph","attrs":{"id":"63377bbe-23bd-45dc-a547-c3b5e5c98199","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23."}]},{"type":"paragraph","attrs":{"id":"cda94ed5-49bf-4a08-9788-1b7f93819478","class":"MsoBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" "}]}]}53:T3e5e,Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation Dongsheng Luo & Efstathios S. Giotis School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK 1. Abstract Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3]. Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii, and Plecotus auritus, representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling (Fig.1 ). We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing. Fig.1 Integrated workflow 2. Introduction Background and rationale Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies. The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance. This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project (https://bat1k.com) and the BatProtect consortium (https://cordis.europa.eu/project/id/101118919). The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses. Hypotheses H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species. Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species. H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species. Primary outcome: differentially expressed genes shared across multiple species following stimulation. H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes. Primary outcome: species-specific or differentially regulated genes and pathways following stimulation. H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation. This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility. 3. Methods Participants/Sample This study does not involve human participants or new animal experimentation. The experimental samples consist of established bat cell cultures from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii and Plecotus auritus. Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design. All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards. Experimental Design and Sequencing Metrics The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing. For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2: Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples Sequencing Specifications: • Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system). • Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp). • Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample. • Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing. Materials and Procedures Work Package 1: RNA quality control and validation of innate immune activation Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction. Work Package 2: Comparative RNA sequencing of bat innate immune responses All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek® Flow® software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant. The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow: 1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment. 2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium. 3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow. Work Package 3: Discovery and prioritisation of candidate antiviral ISGs Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into: • Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages. • Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis). • Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation. Package 4: Data integration and dissemination The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes. Ethics and Data Management The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted. 4. Pilot Data (Ongoing Preliminary Experiment) The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway. The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions. This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study. 5. Work Packages and Timeline Project duration: 9 months Work Package / Activity M1 M2 M3 M4 M5 M6 M7 M8 M9 WP1: RNA QC and qPCR validation ■ ■ WP2: RNA-seq & Partek Flow bioinformatics ■ ■ ■ ■ WP3: ISG discovery, Bat1K analysis ■ ■ ■ WP4: Integration and dissemination ■ ■ Key milestones Milestone Description Target MS1 RNA QC and innate immune response validation completed End of Month 2 MS2 RNA sequencing completed or underway End of Month 3 MS3 Species-specific differential expression analysis completed End of Month 5 MS4 Candidate ISGs prioritised and key findings integrated End of Month 7 MS5 Dataset and analysis outputs prepared for dissemination End of Month 9 6. Budget Proposed budget: $10,000 Budget Item Details & Justification Estimated Cost (USD) RNA Quality Control & Sample Prep Bioanalyzer chips, NanoDrop consumables, quantification kits $400 RT-qPCR Validation Reagents cDNA synthesis kits, SYBR Green master mixes, validated bat primers $1,200 RNA-seq Library Preparation Stranded mRNA library preparation kits for 24 samples $2,000 RNA Sequencing (NovaSeq PE150) Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files) $5,000 Targeted Validation & Functional Reagents Expanded independent RT-qPCR validation assays and molecular reagents $900 General Laboratory Consumables Pipette tips, nuclease-free plasticware, cold-chain reagents $500 Bioinformatics & Partek Flow® Licences Covered by existing lab infrastructure & institutional compute $0 Total $10,000 Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek® Flow® software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding). 7. Expected Impact This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design. 8. References 1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192. 2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214. 3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113. 4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0. 5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23. 54:T1486a,{"type":"doc","content":[{"type":"heading","attrs":{"id":"3114926e-7a4f-41c6-8ba7-5b8ee1ef0335","data-toc-id":"3114926e-7a4f-41c6-8ba7-5b8ee1ef0335","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"18pt","fontFamily":"","color":"black"}},{"type":"bold"}],"text":"Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation"}]},{"type":"heading","attrs":{"id":"64e5a1a5-e226-4d91-985c-73644993f9e2","data-toc-id":"64e5a1a5-e226-4d91-985c-73644993f9e2","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Dongsheng Luo & Efstathios S. Giotis"}]},{"type":"paragraph","attrs":{"id":"3b5ce86e-a4e2-47ba-8b02-949a86c58e8a","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":""}}],"text":"School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK"}]},{"type":"heading","attrs":{"id":"1332fbc7-5d7d-43d7-92b8-83cfa8cd8350","data-toc-id":"1332fbc7-5d7d-43d7-92b8-83cfa8cd8350","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"1. Abstract"}]},{"type":"paragraph","attrs":{"id":"26e2087b-3865-4a9a-b5f5-0b637ec302d3","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3]."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Myotis myotis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Rhinolophus ferrumequinum"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Myotis bechsteinii"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Plecotus auritus"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling ("},{"type":"text","marks":[{"type":"bold"}],"text":"Fig.1"},{"type":"text","text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":")."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing."}]},{"type":"imageBlock","attrs":{"src":"https://storage.prod.researchhub.com/uploads/notes/users/207668/7f21793a-2fd8-4604-8f5d-4b23da25077d/image.png","width":"100%","align":"left"}},{"type":"paragraph","attrs":{"id":"1d751b3f-b77c-4117-aa22-3b41e5c16877","class":null,"textAlign":"center"},"content":[{"type":"text","marks":[{"type":"bold"}],"text":"Fig.1"},{"type":"text","text":" Integrated workflow"}]},{"type":"heading","attrs":{"id":"94b7fb54-9fea-4faf-9234-e7ad13fd8f1a","data-toc-id":"94b7fb54-9fea-4faf-9234-e7ad13fd8f1a","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"2. Introduction"}]},{"type":"heading","attrs":{"id":"228101a2-90c2-4b2a-ab7e-a92b3772625b","data-toc-id":"228101a2-90c2-4b2a-ab7e-a92b3772625b","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Background and rationale"}]},{"type":"paragraph","attrs":{"id":"9ffad870-a4ce-457f-9a23-7b9fe9b82fc6","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance."}]},{"type":"paragraph","attrs":{"id":"48112859-fa4c-49b8-b3ab-65f1e73f69c0","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project ("},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://bat1k.com","target":"_blank","rel":"noopener noreferrer nofollow","class":"link","title":null}},{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":""}},{"type":"underline"}],"text":"https://bat1k.com"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":") and the BatProtect consortium ("},{"type":"text","marks":[{"type":"link","attrs":{"href":"https://cordis.europa.eu/project/id/101118919","target":"_blank","rel":"noopener noreferrer nofollow","class":"link","title":null}},{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"blue"}},{"type":"underline"}],"text":"https://cordis.europa.eu/project/id/101118919"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":")."}]},{"type":"paragraph","attrs":{"id":"21f3b9f4-f371-4ccd-9fdd-665ffa70bfe8","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses."}]},{"type":"heading","attrs":{"id":"61c26901-7322-4452-8ab9-28b247bd813a","data-toc-id":"61c26901-7322-4452-8ab9-28b247bd813a","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Hypotheses"}]},{"type":"heading","attrs":{"id":"8d741253-4483-43fb-92c8-192da4bfac63","data-toc-id":"8d741253-4483-43fb-92c8-192da4bfac63","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species."}]},{"type":"paragraph","attrs":{"id":"1a393cc3-4621-4459-9669-b3c7db7c78ec","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species."}]},{"type":"heading","attrs":{"id":"ebd0b3ca-42fa-4020-bb08-24aa041a784b","data-toc-id":"ebd0b3ca-42fa-4020-bb08-24aa041a784b","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species."}]},{"type":"paragraph","attrs":{"id":"1498c99c-6847-4270-9448-fd133c45936f","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Primary outcome: differentially expressed genes shared across multiple species following stimulation."}]},{"type":"heading","attrs":{"id":"094888d1-a741-4ff8-ace3-7d45a96277ee","data-toc-id":"094888d1-a741-4ff8-ace3-7d45a96277ee","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes."}]},{"type":"paragraph","attrs":{"id":"fae98a17-c0a6-4173-bc8a-2d629c2aa860","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Primary outcome: species-specific or differentially regulated genes and pathways following stimulation."}]},{"type":"heading","attrs":{"id":"b1bd33a5-4060-43dc-9fe0-f49ca17159a6","data-toc-id":"b1bd33a5-4060-43dc-9fe0-f49ca17159a6","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation."}]},{"type":"paragraph","attrs":{"id":"3b4627cc-dc76-453c-a98b-26e324c316de","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility."}]},{"type":"heading","attrs":{"id":"cd09551c-c2d7-4ab5-8fe7-a433828a5c08","data-toc-id":"cd09551c-c2d7-4ab5-8fe7-a433828a5c08","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"3. Methods"}]},{"type":"heading","attrs":{"id":"93dd2c13-0440-4428-bd5a-0ddde6a91a48","data-toc-id":"93dd2c13-0440-4428-bd5a-0ddde6a91a48","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Participants/Sample"}]},{"type":"paragraph","attrs":{"id":"afcd77eb-1fbb-4b5f-b3c1-98ee02f6d09d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This study does not involve human participants or new animal experimentation."}]},{"type":"paragraph","attrs":{"id":"af9406f0-ccc8-4ed8-ba04-83369dd21815","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The experimental samples consist of established bat cell cultures from four European bat species: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Myotis myotis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":"Rhinolophus ferrumequinum, Myotis bechsteinii "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"and"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"italic"}],"text":" Plecotus auritus"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":". Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design."}]},{"type":"paragraph","attrs":{"id":"71359f18-0fdc-4260-a268-20f00cad6c03","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards."}]},{"type":"heading","attrs":{"id":"2d63fe3d-9684-420e-b7df-1b9e1e19701b","data-toc-id":"2d63fe3d-9684-420e-b7df-1b9e1e19701b","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Experimental Design and Sequencing Metrics"}]},{"type":"paragraph","attrs":{"id":"1f97f8cc-e570-4e45-a4d6-2c28cd9fd562","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing."}]},{"type":"paragraph","attrs":{"id":"5a9ae4f0-4437-45d2-93e3-f62849e6e188","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2:"}]},{"type":"paragraph","attrs":{"id":"f92f9332-71dc-4ba8-b45d-dc00b5113206","class":"MsoNormal","textAlign":"center"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}},{"type":"bold"}],"text":"Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples"}]},{"type":"paragraph","attrs":{"id":"1b4fd621-187e-4cad-bc06-f2de626e8948","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Sequencing Specifications:"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system)."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp)."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing."}]},{"type":"paragraph","attrs":{"id":"78aafbb2-b390-41f4-98d1-b8da4859bc0d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"11pt","fontFamily":"","color":"black"}}],"text":" "}]},{"type":"heading","attrs":{"id":"7d729a67-c1a3-47ac-9b34-38e65953e2a4","data-toc-id":"7d729a67-c1a3-47ac-9b34-38e65953e2a4","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Materials and Procedures"}]},{"type":"heading","attrs":{"id":"0be9bdf4-3993-4c98-a4a9-36c9350fd866","data-toc-id":"0be9bdf4-3993-4c98-a4a9-36c9350fd866","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Work Package 1: RNA quality control and validation of innate immune activation"}]},{"type":"paragraph","attrs":{"id":"77195db0-31ad-4569-b498-eca55a5bde8c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction."}]},{"type":"heading","attrs":{"id":"df923cef-33a0-457a-8b22-5accf066d9e9","data-toc-id":"df923cef-33a0-457a-8b22-5accf066d9e9","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Work Package 2: Comparative RNA sequencing of bat innate immune responses"}]},{"type":"paragraph","attrs":{"id":"17760ed7-effb-489c-99a0-581e2bf79e6f","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" Flow"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant."}]},{"type":"paragraph","attrs":{"id":"36d48ef8-4918-4baf-a6de-2552c90e6915","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow:"}]},{"type":"paragraph","attrs":{"id":"41d6e956-5ed9-4da7-a6f6-846a5b71b9cd","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment."}]},{"type":"paragraph","attrs":{"id":"83a822a7-4263-46dd-8fd2-a11c852ff703","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium."}]},{"type":"paragraph","attrs":{"id":"bd9a8815-4ee8-4409-ac8c-91e4ff61d743","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow."}]},{"type":"heading","attrs":{"id":"1bae9cbb-05d2-4bfe-af59-143eb5a1a5f6","data-toc-id":"1bae9cbb-05d2-4bfe-af59-143eb5a1a5f6","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Work Package 3: Discovery and prioritisation of candidate antiviral ISGs"}]},{"type":"paragraph","attrs":{"id":"c215bb45-9289-4dc7-8821-ab363f9368e4","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into:"}]},{"type":"paragraph","attrs":{"id":"760432e6-facd-4474-886c-a6cab33fada7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages."}]},{"type":"paragraph","attrs":{"id":"b81b8a79-4372-4df4-abc9-936ea3a1211e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis)."}]},{"type":"paragraph","attrs":{"id":"ab7d97dc-4856-47c8-98fd-29f25403d725","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"• Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation."}]},{"type":"heading","attrs":{"id":"670a48ca-3530-4998-96c5-2ce4a6436a5c","data-toc-id":"670a48ca-3530-4998-96c5-2ce4a6436a5c","textAlign":null,"level":3},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Package 4: Data integration and dissemination"}]},{"type":"paragraph","attrs":{"id":"1645997c-2e3d-48f2-9796-cd1aa9a25f75","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes."}]},{"type":"paragraph","attrs":{"id":"e1a00751-d424-417f-98e2-2876cfb62cf2","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":" "}]},{"type":"heading","attrs":{"id":"83b69c0c-3ca4-42de-b40f-024b21e847f4","data-toc-id":"83b69c0c-3ca4-42de-b40f-024b21e847f4","textAlign":null,"level":2},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"Ethics and Data Management"}]},{"type":"paragraph","attrs":{"id":"43348c2c-60eb-4153-8f6d-cbab2e314ccb","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted."}]},{"type":"heading","attrs":{"id":"5e03900a-e596-4017-b699-371413e262fc","data-toc-id":"5e03900a-e596-4017-b699-371413e262fc","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"4. Pilot Data (Ongoing Preliminary Experiment)"}]},{"type":"paragraph","attrs":{"id":"a87f12ed-46e0-4c5b-907d-547c4e958981","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions."},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study."}]},{"type":"heading","attrs":{"id":"e1bad060-0b6a-46e5-923b-b0ce175825dd","data-toc-id":"e1bad060-0b6a-46e5-923b-b0ce175825dd","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"5. Work Packages and Timeline"}]},{"type":"paragraph","attrs":{"id":"b5cc5d33-8d04-43d2-bbee-50df3dd2fdf7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Project duration: 9 months"}]},{"type":"table","attrs":{"id":"f6e9f1a8-fa9d-4918-8281-f5f6956f5054"},"content":[{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":[156],"style":"width: 195.2pt; border: 1pt solid windowtext; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 25.9pt;"},"content":[{"type":"paragraph","attrs":{"id":"c91159c7-3bfc-48a0-a2b3-84646afe56e8","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Work Package / Activity"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":[25],"style":"width: 26.1pt; border-width: 1pt 1pt 1pt medium; 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border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt;"},"content":[{"type":"paragraph","attrs":{"id":"0d788675-8294-408f-9b45-ae5e69bf6572","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Dataset and analysis outputs prepared for dissemination"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 108.25pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt;"},"content":[{"type":"paragraph","attrs":{"id":"99706d5c-a614-44a1-98dd-c75f84b71b13","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"End of Month 9"}]}]}]}]},{"type":"heading","attrs":{"id":"f83650ff-8d89-4b46-b579-b5c1f435503e","data-toc-id":"f83650ff-8d89-4b46-b579-b5c1f435503e","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"6. Budget"}]},{"type":"paragraph","attrs":{"id":"64f44e46-63dc-46f7-837e-6dbf5f93b7cf","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Proposed budget: $10,000"}]},{"type":"table","attrs":{"id":"0134fb1c-fc2c-44fb-af27-cc46a733b974"},"content":[{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border: 1pt solid windowtext; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 9.65pt;"},"content":[{"type":"paragraph","attrs":{"id":"b5b2ae91-4df2-45ad-8b7d-6cce2b68eade","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Budget Item"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-color: windowtext windowtext windowtext currentcolor; border-image: none; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 9.65pt;"},"content":[{"type":"paragraph","attrs":{"id":"88112b04-034c-4896-b988-b1a8d1483d36","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Details & Justification"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-color: windowtext windowtext windowtext currentcolor; border-image: none; background: rgb(217, 217, 217); padding: 0cm 5.4pt; height: 9.65pt;"},"content":[{"type":"paragraph","attrs":{"id":"d3a0f446-ae20-4ff1-b4c8-3923ab50511b","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"","color":"black"}}],"text":"Estimated Cost (USD)"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; 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border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"8a42681c-3d29-4c43-8f87-8d9f274186a7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$400"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"d480c40f-66ba-4462-a8b5-b2c772b80d8e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RT-qPCR Validation Reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"010bbec9-fd19-4741-9f91-7785475f6082","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"cDNA synthesis kits, SYBR Green master mixes, validated bat primers"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"e1a22ebc-3558-471e-a370-c5101d902a9c","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$1,200"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"7bd0bd3e-65f6-408b-824b-219ef56641c5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RNA-seq Library Preparation"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"a495180b-623c-49f1-8162-ce4b32e6f049","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Stranded mRNA library preparation kits for 24 samples"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"328090ad-585d-443e-a616-f0a961c4d8a6","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$2,000"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 38.75pt;"},"content":[{"type":"paragraph","attrs":{"id":"1aff34b1-e286-4634-83f3-324ce04bcf0a","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"RNA Sequencing (NovaSeq PE150)"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 38.75pt;"},"content":[{"type":"paragraph","attrs":{"id":"8a0828e5-b61c-4097-a651-fb58da83a700","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files)"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 38.75pt;"},"content":[{"type":"paragraph","attrs":{"id":"01dfc661-f390-4c9e-846a-c56b312372e9","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$5,000"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"add3741f-0190-41e6-a63d-ef627852cbb7","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Targeted Validation & Functional Reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"28637b8c-1b51-47ca-a0d6-0a529917c968","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Expanded independent RT-qPCR validation assays and molecular reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"173e0792-d5ab-499e-91bd-801a2e66e1d1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$900 "}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.7pt;"},"content":[{"type":"paragraph","attrs":{"id":"e561a617-c808-4573-902c-65ec5dbfbb7d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"General Laboratory Consumables"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.7pt;"},"content":[{"type":"paragraph","attrs":{"id":"1dd50f80-88b3-489e-88de-4abef7d86ef5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Pipette tips, nuclease-free plasticware, cold-chain reagents"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.7pt;"},"content":[{"type":"paragraph","attrs":{"id":"e93951c8-cfa5-40da-9e94-a437f6f26caf","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$500"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"ad6f8c74-1598-4a24-b6eb-52580facb014","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Bioinformatics & Partek Flow"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","text":" Licences"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"0a6b0c51-35d7-43ac-8e6f-1e6fa533291e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Covered by existing lab infrastructure & institutional compute"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 29.05pt;"},"content":[{"type":"paragraph","attrs":{"id":"8cc96634-a2ac-495c-a60f-0003598090b3","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$0"}]}]}]},{"type":"tableRow","content":[{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 168.2pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-color: currentcolor windowtext windowtext; border-image: none; padding: 0cm 5.4pt; height: 21.25pt;"},"content":[{"type":"paragraph","attrs":{"id":"5823d16e-3e57-4b2f-bf56-3dcce2f94b10","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"Total"}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 198.7pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 21.25pt;"},"content":[{"type":"paragraph","attrs":{"id":"bb024584-8505-456d-86cc-24cba28b6696","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":" "}]}]},{"type":"tableCell","attrs":{"colspan":1,"rowspan":1,"colwidth":null,"style":"width: 92.1pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; border-color: currentcolor windowtext windowtext currentcolor; padding: 0cm 5.4pt; height: 21.25pt;"},"content":[{"type":"paragraph","attrs":{"id":"01fc23a8-3f66-4514-b3d0-7ce1449f0af5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","text":"$10,000"}]}]}]}]},{"type":"paragraph","attrs":{"id":"a226e565-52f4-4781-bb01-d8eff254fbc8","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" Flow"},{"type":"emoji","attrs":{"name":"registered"}},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding)."}]},{"type":"heading","attrs":{"id":"8482e1ad-bb6e-4f05-a0ed-d5ab1fdd8644","data-toc-id":"8482e1ad-bb6e-4f05-a0ed-d5ab1fdd8644","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"7. Expected Impact"}]},{"type":"paragraph","attrs":{"id":"d1de65d2-d420-42a9-92ec-e7274ae347e5","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design."}]},{"type":"heading","attrs":{"id":"8b09d553-9d07-4ba4-95e6-e68bd43b6e8b","data-toc-id":"8b09d553-9d07-4ba4-95e6-e68bd43b6e8b","textAlign":null,"level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"Arial, sans-serif","color":"black"}}],"text":"8. References"}]},{"type":"paragraph","attrs":{"id":"e5a4cbd2-b808-4d8d-8300-f91cd1926a26","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192."}]},{"type":"paragraph","attrs":{"id":"7bf55c4c-9f14-41dd-89e7-9ced36f8b81d","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214."}]},{"type":"paragraph","attrs":{"id":"13b9bd7d-9915-4823-9062-90cf325510b1","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113."}]},{"type":"paragraph","attrs":{"id":"37f96245-b1ef-442d-9950-006fe5afd75a","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0."}]},{"type":"paragraph","attrs":{"id":"63377bbe-23bd-45dc-a547-c3b5e5c98199","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":"5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23."}]},{"type":"paragraph","attrs":{"id":"cda94ed5-49bf-4a08-9788-1b7f93819478","class":"MsoBibliography","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"12pt","fontFamily":"","color":"black"}}],"text":" "}]}]}55:Te735,

Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation

Dongsheng Luo & Efstathios S. Giotis

School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK

1. Abstract

Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3].

Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii, and Plecotus auritus, representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling (Fig.1 ).

We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing.

Fig.1 Integrated workflow

2. Introduction

Background and rationale

Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies.

The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance.

This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project (https://bat1k.com) and the BatProtect consortium (https://cordis.europa.eu/project/id/101118919).

The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses.

Hypotheses

H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species.

Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species.

H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species.

Primary outcome: differentially expressed genes shared across multiple species following stimulation.

H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes.

Primary outcome: species-specific or differentially regulated genes and pathways following stimulation.

H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation.

This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility.

3. Methods

Participants/Sample

This study does not involve human participants or new animal experimentation.

The experimental samples consist of established bat cell cultures from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii and Plecotus auritus. Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design.

All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards.

Experimental Design and Sequencing Metrics

The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing.

For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2:

Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples

Sequencing Specifications:
• Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system).
• Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp).
• Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample.
• Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing.

Materials and Procedures

Work Package 1: RNA quality control and validation of innate immune activation

Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction.

Work Package 2: Comparative RNA sequencing of bat innate immune responses

All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek® Flow® software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant.

The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow:

1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment.

2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium.

3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow.

Work Package 3: Discovery and prioritisation of candidate antiviral ISGs

Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into:

• Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages.

• Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis).

• Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation.

Package 4: Data integration and dissemination

The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes.

Ethics and Data Management

The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted.

4. Pilot Data (Ongoing Preliminary Experiment)

The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway.

The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions.

This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study.

5. Work Packages and Timeline

Project duration: 9 months

Work Package / Activity	M1	M2	M3	M4	M5	M6	M7	M8	M9
WP1: RNA QC and qPCR validation	■	■
WP2: RNA-seq & Partek Flow bioinformatics		■	■	■	■
WP3: ISG discovery, Bat1K analysis					■	■	■
WP4: Integration and dissemination								■	■

Key milestones

Milestone	Description	Target
MS1	RNA QC and innate immune response validation completed	End of Month 2
MS2	RNA sequencing completed or underway	End of Month 3
MS3	Species-specific differential expression analysis completed	End of Month 5
MS4	Candidate ISGs prioritised and key findings integrated	End of Month 7
MS5	Dataset and analysis outputs prepared for dissemination	End of Month 9

6. Budget

Proposed budget: $10,000

Budget Item	Details & Justification	Estimated Cost (USD)
RNA Quality Control & Sample Prep	Bioanalyzer chips, NanoDrop consumables, quantification kits	$400
RT-qPCR Validation Reagents	cDNA synthesis kits, SYBR Green master mixes, validated bat primers	$1,200
RNA-seq Library Preparation	Stranded mRNA library preparation kits for 24 samples	$2,000
RNA Sequencing (NovaSeq PE150)	Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files)	$5,000
Targeted Validation & Functional Reagents	Expanded independent RT-qPCR validation assays and molecular reagents	$900
General Laboratory Consumables	Pipette tips, nuclease-free plasticware, cold-chain reagents	$500
Bioinformatics & Partek Flow® Licences	Covered by existing lab infrastructure & institutional compute	$0
Total		$10,000

Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek® Flow® software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding).

7. Expected Impact

This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design.

8. References

1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192.

2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214.

3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113.

4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0.

5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23.

56:Te735,

Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation

Dongsheng Luo & Efstathios S. Giotis

School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK

1. Abstract

Fig.1 Integrated workflow

2. Introduction

Background and rationale

Hypotheses

H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species.

Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species.

H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species.

Primary outcome: differentially expressed genes shared across multiple species following stimulation.

H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes.

Primary outcome: species-specific or differentially regulated genes and pathways following stimulation.

H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation.

This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility.

3. Methods

Participants/Sample

This study does not involve human participants or new animal experimentation.

Experimental Design and Sequencing Metrics

Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples

Materials and Procedures

Work Package 1: RNA quality control and validation of innate immune activation

Work Package 2: Comparative RNA sequencing of bat innate immune responses

The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow:

1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment.

2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium.

3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow.

Work Package 3: Discovery and prioritisation of candidate antiviral ISGs

Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into:

• Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages.

• Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis).

• Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation.

Package 4: Data integration and dissemination

Ethics and Data Management

4. Pilot Data (Ongoing Preliminary Experiment)

5. Work Packages and Timeline

Project duration: 9 months

Work Package / Activity	M1	M2	M3	M4	M5	M6	M7	M8	M9
WP1: RNA QC and qPCR validation	■	■
WP2: RNA-seq & Partek Flow bioinformatics		■	■	■	■
WP3: ISG discovery, Bat1K analysis					■	■	■
WP4: Integration and dissemination								■	■

Key milestones

Milestone	Description	Target
MS1	RNA QC and innate immune response validation completed	End of Month 2
MS2	RNA sequencing completed or underway	End of Month 3
MS3	Species-specific differential expression analysis completed	End of Month 5
MS4	Candidate ISGs prioritised and key findings integrated	End of Month 7
MS5	Dataset and analysis outputs prepared for dissemination	End of Month 9

6. Budget

Proposed budget: $10,000

Budget Item	Details & Justification	Estimated Cost (USD)
RNA Quality Control & Sample Prep	Bioanalyzer chips, NanoDrop consumables, quantification kits	$400
RT-qPCR Validation Reagents	cDNA synthesis kits, SYBR Green master mixes, validated bat primers	$1,200
RNA-seq Library Preparation	Stranded mRNA library preparation kits for 24 samples	$2,000
RNA Sequencing (NovaSeq PE150)	Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files)	$5,000
Targeted Validation & Functional Reagents	Expanded independent RT-qPCR validation assays and molecular reagents	$900
General Laboratory Consumables	Pipette tips, nuclease-free plasticware, cold-chain reagents	$500
Bioinformatics & Partek Flow® Licences	Covered by existing lab infrastructure & institutional compute	$0
Total		$10,000

7. Expected Impact

8. References

1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192.

4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0.

57:T3e5a,Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation Dongsheng Luo & Efstathios S. Giotis School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK 1. Abstract Bats harbour a remarkable diversity of viruses while often displaying limited disease, suggesting that their antiviral immune systems have evolved mechanisms that balance efficient virus control with restricted immunopathology [1]. Interferon-stimulated genes (ISGs) are central effectors of innate antiviral immunity, but the diversity, conservation and functional significance of ISG responses across bat species remain poorly understood [2,3]. Building upon foundational platforms and technology established through our current Medical Research Council (MRC)-funded research on bat innate immunity, our lab has established an active collaboration with Dr. Laurent Dacheux (Institut Pasteur, Paris). Through this collaboration, we have secured established bat cells from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii, and Plecotus auritus, representing distinct evolutionary lineages, fully covered under an executed Material Transfer Agreement (MTA) addressing all legal and regulatory requirements. Using synthetic 5′-triphosphorylated RNA (5′ppp RNA Lyovec) to stimulate RIG-I-associated innate immune signalling, we are generating a comparative dataset of antiviral responses across these species. The stimulation experiment and RNA extraction have been completed. We will first validate innate immune activation by targeted RT-qPCR and subsequently perform paired-end RNA sequencing (Illumina NovaSeq PE150) across 24 biological samples. Downstream processing of all 48 raw FASTQ files will be conducted using our existing high-performance bioinformatics infrastructure and established expertise in Partek Flow (R), integrated with genomic datasets from the Bat1K and BatProtect consortia through collaborations with Prof. Linfa Wang and Prof. Emma Teeling (Fig.1 ). We hypothesise that comparative transcriptomics will identify bat-specific or differentially regulated candidate ISGs associated with effective antiviral defence. This nine-month project will generate a comparative resource and prioritise candidate antiviral genes for future functional investigation. By fully leveraging existing compute resources and Partek Flow software licences, project funding is focused entirely on wet-lab validation and sequencing. Fig.1 Integrated workflow 2. Introduction Background and rationale Bats are important hosts of a remarkable diversity of viruses, yet many bat species appear able to tolerate viral infections without the severe pathology observed in other mammals [4]. Understanding the molecular mechanisms underlying this phenotype could reveal fundamental principles of antiviral immunity and identify new pathways relevant to host-directed antiviral strategies. The innate immune response provides the first line of defence against RNA viruses. Cytoplasmic viral RNA can be detected by pattern-recognition receptors such as retinoic acid-inducible gene I (RIG-I), leading to downstream signalling and induction of interferons and interferon-stimulated genes (ISGs) [5]. Although innate immune pathways have been investigated in individual bat species, it remains unclear to what extent antiviral ISG responses are conserved across bats and whether different species possess distinctive transcriptional programmes that may contribute to viral tolerance. This project capitalises on our ongoing MRC-funded research program in the Giotis laboratory, which has established the experimental baseline, cell model pipelines, and RIG-I stimulation protocols. Furthermore, through our strategic collaboration with Dr. Laurent Dacheux at the Institut Pasteur (Paris), we have acquired four unique, well-characterised European bat cell lines under formal MTAs. To maximise the biological and evolutionary interpretation of these datasets, our framework links directly with leading international bat genomics consortia through ongoing collaborations with Prof. Linfa Wang (Duke-NUS) and Prof. Emma Teeling (University College Dublin), leveraging reference genomes and comparative immunogenomic data from the Bat1K genome project (https://bat1k.com) and the BatProtect consortium (https://cordis.europa.eu/project/id/101118919). The experiment is now at the RT-qPCR stage, with high-throughput RNA sequencing planned as the next phase. This project directly expands upon these ongoing experiments to generate a comparative map of RIG-I-associated antiviral transcriptional responses. Hypotheses H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species. Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species. H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species. Primary outcome: differentially expressed genes shared across multiple species following stimulation. H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes. Primary outcome: species-specific or differentially regulated genes and pathways following stimulation. H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation. This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility. 3. Methods Participants/Sample This study does not involve human participants or new animal experimentation. The experimental samples consist of established bat cell cultures from four European bat species: Myotis myotis, Rhinolophus ferrumequinum, Myotis bechsteinii and Plecotus auritus. Cells have been challenged with 5′ppp RNA (Lyovec) using the ongoing experimental protocol. The subsequent analyses will compare stimulated samples with appropriate experimental controls included in the existing study design. All cell lines were obtained from Dr. Laurent Dacheux under fully executed Material Transfer Agreements (MTAs), ensuring compliance with institutional, national, and international legal and biosafety standards. Experimental Design and Sequencing Metrics The study employs a robust biological replication framework for transcriptomic discovery. The human A549 cell line will be included alongside the bat species in the initial RT-qPCR validation phase (WP1) as a mammalian positive control to confirm experimental RIG-I pathway activation. However, to direct sequencing resources entirely towards novel bat ISG discovery, A549 cells will be excluded from the RNA sequencing. For the RNA sequencing cohort, cells from each of the four bat species are allocated into two experimental conditions (5′ppp RNA (Lyovec) -stimulated vs. mock vehicle control) in triplicate. Maintaining three biological replicates per condition is essential to calculate biological variance and provide sufficient statistical power for differential expression analysis using DESeq2: Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples Sequencing Specifications: • Platform: Illumina NovaSeq 6000 / NovaSeq X platform (or equivalent high-throughput short-read system). • Library Type: Stranded paired-end mRNA sequencing (PE150, 2 × 150 bp). • Sequencing Depth: Targeted minimum of 30–40 million read pairs (60–80 million total reads) per sample. • Raw Data Volume: 24 paired-end libraries yielding 48 raw FASTQ files for downstream bioinformatic processing. Materials and Procedures Work Package 1: RNA quality control and validation of innate immune activation Total RNA extraction is completed. RNA integrity will be evaluated using an Agilent 2100 Bioanalyzer (requiring RIN ≥ 8.0). Targeted RT-qPCR panels will quantify induction of canonical innate immune markers (e.g., IFNB1, IFIT1, ISG15, OAS1, MX1, DDX58 and CXCL10) to validate RIG-I activation prior to sequencing library construction. Work Package 2: Comparative RNA sequencing of bat innate immune responses All bioinformatics analysis will be conducted using the Giotis lab's dedicated, pre-funded high-performance computing infrastructure and commercial Partek® Flow® software licences. By utilising our existing Partek Flow deployment and in-house expertise, we achieve streamlined, reproducible data processing without incurring bioinformatics overhead on this grant. The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow: 1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment. 2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium. 3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow. Work Package 3: Discovery and prioritisation of candidate antiviral ISGs Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into: • Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages. • Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis). • Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation. Package 4: Data integration and dissemination The final phase will integrate results into a comparative resource describing RIG-I-associated transcriptional responses across the four bat species. Processed data, metadata, protocols and analysis code will be prepared for open sharing where appropriate. Results will also provide the basis for a manuscript, preprint or future functional studies of prioritised candidate genes. Ethics and Data Management The proposed work uses established bat-derived cell cultures and does not involve new animal sampling, animal experimentation or human participants. All work will be conducted under applicable institutional biosafety, ethical and data-management requirements. Experimental data will be stored on secure institutional systems. Processed datasets, relevant metadata, protocols and analysis code will be shared through appropriate public repositories where permitted. 4. Pilot Data (Ongoing Preliminary Experiment) The proposal is supported by an ongoing preliminary experiment using bat cell cultures from four European species challenged with 5′ppp RNA to stimulate RIG-I-associated innate immune signalling. The stimulation experiment has been completed, and RNA extraction is currently underway. The immediate next steps are targeted RT-qPCR to confirm and characterise the innate immune response, followed by RNA sequencing for discovery of broader transcriptional changes. At the time of submission, the pilot data are therefore evidence of experimental feasibility and project progress rather than definitive biological conclusions. This distinction is important: the proposal does not claim that novel ISGs have already been discovered. Instead, it uses the ongoing experiment as a direct and feasible foundation for the proposed comparative transcriptomic study. 5. Work Packages and Timeline Project duration: 9 months Work Package / Activity M1 M2 M3 M4 M5 M6 M7 M8 M9 WP1: RNA QC and qPCR validation ■ ■ WP2: RNA-seq & Partek Flow bioinformatics ■ ■ ■ ■ WP3: ISG discovery, Bat1K analysis ■ ■ ■ WP4: Integration and dissemination ■ ■ Key milestones Milestone Description Target MS1 RNA QC and innate immune response validation completed End of Month 2 MS2 RNA sequencing completed or underway End of Month 3 MS3 Species-specific differential expression analysis completed End of Month 5 MS4 Candidate ISGs prioritised and key findings integrated End of Month 7 MS5 Dataset and analysis outputs prepared for dissemination End of Month 9 6. Budget Proposed budget: $10,000 Budget Item Details & Justification Estimated Cost (USD) RNA Quality Control & Sample Prep Bioanalyzer chips, NanoDrop consumables, quantification kits $400 RT-qPCR Validation Reagents cDNA synthesis kits, SYBR Green master mixes, validated bat primers $1,200 RNA-seq Library Preparation Stranded mRNA library preparation kits for 24 samples $2,000 RNA Sequencing (NovaSeq PE150) Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files) $5,000 Targeted Validation & Functional Reagents Expanded independent RT-qPCR validation assays and molecular reagents $900 General Laboratory Consumables Pipette tips, nuclease-free plasticware, cold-chain reagents $500 Bioinformatics & Partek Flow® Licences Covered by existing lab infrastructure & institutional compute $0 Total $10,000 Budget Justification Note: To maximize cost-efficiency and direct grant funding toward wet-lab generation of high-value experimental data, all computational infrastructure, storage, and Partek® Flow® software licensing costs are fully covered by existing departmental resources and ongoing Giotis lab infrastructure (supported in part by current MRC funding). 7. Expected Impact This project addresses a major knowledge gap in comparative bat virology and immunology. By integrating unique, MTA-cleared European bat cell models (Institut Pasteur) with high-depth transcriptomics and the reference frameworks of the Bat1K and BatProtect initiatives (Prof. Linfa Wang, Prof. Emma Teeling), this study establishes a scalable comparative platform for bat antiviral discovery. The resulting datasets will provide a foundation for identifying species-agnostic pan-viral restriction factors and therapeutic targets for host-directed antiviral design. 8. References 1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192. 2. De La Cruz-Rivera, P.C.; Kanchwala, M.; Liang, H.; Kumar, A.; Wang, L.-F.; Xing, C.; Schoggins, J.W. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. The Journal of Immunology 2018, 200, 209–217, doi:10.4049/jimmunol.1701214. 3. Zhou, P.; Tachedjian, M.; Wynne, J.W.; Boyd, V.; Cui, J.; Smith, I.; Cowled, C.; Ng, J.H.J.; Mok, L.; Michalski, W.P.; et al. Contraction of the Type I IFN Locus and Unusual Constitutive Expression of IFN-α in Bats. Proceedings of the National Academy of Sciences 2016, 113, 2696–2701, doi:10.1073/pnas.1518240113. 4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0. 5. Schoen Andreas; Hölzer Martin; Müller Marcel A.; Wallerang Kai B.; Drosten Christian; Marz Manja; Lamp Benjamin; Weber Friedemann Functional Comparisons of the Virus Sensor RIG-I from Humans, the Microbat Myotis Daubentonii, and the Megabat Rousettus Aegyptiacus, and Their Response to SARS-CoV-2 Infection. Journal of Virology 2023, 97, e00205-23, doi:10.1128/jvi.00205-23.20:["$","$L4e",null,{"children":["$","$L4",null,{"topBanner":["$","$L4f",null,{"work":{"id":32853,"type":"$undefined","contentType":"preregistration","title":"Comparative Discovery of Antiviral ISGs in European Bats Following RIG-I Activation","slug":"comparative-discovery-of-antiviral-isgs-in-european-bats-following-rig-i-activation","createdDate":"2026-08-31T16:54:11.864301Z","createdByUserId":207668,"updatedDate":"2026-08-31T17:16:30.263024Z","publishedDate":"2026-08-31T16:54:11.864301Z","authors":[{"authorProfile":{"id":8713729,"fullName":"Dongsheng Luo","firstName":"Dongsheng","lastName":"Luo","profileImage":"","headline":"Senior Research Officer at University of Essex","profileUrl":"/author/8713729","user":{"id":0,"email":"","firstName":"","lastName":"","fullName":"Unknown 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5c:Ta9a1,

School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK

Fig.1 Integrated workflow

Background and rationale

Hypotheses

H1. RIG-I stimulation induces a measurable innate antiviral transcriptional response in all four bat species.

Primary outcome: differential expression of predefined innate immune and ISG markers following 5′ppp RNA stimulation. Secondary outcome: differences in the magnitude of responses among species.

H2. A core set of antiviral ISGs is conserved across evolutionarily distinct European bat species.

Primary outcome: differentially expressed genes shared across multiple species following stimulation.

H3. Individual bat species exhibit distinct ISG signatures and candidate antiviral genes.

Primary outcome: species-specific or differentially regulated genes and pathways following stimulation.

H4. Comparative transcriptomics can identify previously under-characterised candidate antiviral genes for future functional investigation.

This is an exploratory discovery hypothesis. Candidates will be prioritised based on reproducibility, magnitude of induction, cross-species patterns and biological plausibility.

Participants/Sample

This study does not involve human participants or new animal experimentation.

Experimental Design and Sequencing Metrics

Total Sequencing Sample Count = 4 bat species × 2 conditions (Stimulated vs. Mock) × 3 biological replicates = 24 samples

Materials and Procedures

Work Package 1: RNA quality control and validation of innate immune activation

Work Package 2: Comparative RNA sequencing of bat innate immune responses

The 48 raw FASTQ files will undergo an automated, standardised workflow within Partek Flow:

1. Pre-alignment Quality Control: Read quality scoring, adapter trimming, and base composition assessment.

2. Alignment and Quantification: Spliced alignment using STAR against chromosome-level bat genomes generated by the Bat1K consortium.

3. Differential Expression: Statistical modelling using DESeq2 within Partek Flow.

Work Package 3: Discovery and prioritisation of candidate antiviral ISGs

Differentially expressed gene sets will be cross-referenced across species using databases and high-confidence annotations from the Bat1K project. Genes will be categorised into:

• Conserved Core Bat ISGs: Transcripts uniformly induced across all four lineages.

• Lineage-Specific Effectors: Genes uniquely responsive in specific bat taxa (e.g., Rhinolophus vs. Myotis).

• Novel/Uncharacterised Candidates: Transcripts with divergent regulatory profiles or uncharacterised immune functions, prioritised for independent RT-qPCR validation.

Package 4: Data integration and dissemination

Ethics and Data Management

Project duration: 9 months

Work Package / Activity	M1	M2	M3	M4	M5	M6	M7	M8	M9
WP1: RNA QC and qPCR validation	■	■
WP2: RNA-seq & Partek Flow bioinformatics		■	■	■	■
WP3: ISG discovery, Bat1K analysis					■	■	■
WP4: Integration and dissemination								■	■

Key milestones

Milestone	Description	Target
MS1	RNA QC and innate immune response validation completed	End of Month 2
MS2	RNA sequencing completed or underway	End of Month 3
MS3	Species-specific differential expression analysis completed	End of Month 5
MS4	Candidate ISGs prioritised and key findings integrated	End of Month 7
MS5	Dataset and analysis outputs prepared for dissemination	End of Month 9

Proposed budget: $10,000

Budget Item	Details & Justification	Estimated Cost (USD)
RNA Quality Control & Sample Prep	Bioanalyzer chips, NanoDrop consumables, quantification kits	$400
RT-qPCR Validation Reagents	cDNA synthesis kits, SYBR Green master mixes, validated bat primers	$1,200
RNA-seq Library Preparation	Stranded mRNA library preparation kits for 24 samples	$2,000
RNA Sequencing (NovaSeq PE150)	Paired-end sequencing (2 × 150 bp, 30–40M reads/sample; 48 FASTQ files)	$5,000
Targeted Validation & Functional Reagents	Expanded independent RT-qPCR validation assays and molecular reagents	$900
General Laboratory Consumables	Pipette tips, nuclease-free plasticware, cold-chain reagents	$500
Bioinformatics & Partek Flow® Licences	Covered by existing lab infrastructure & institutional compute	$0
Total		$10,000

1. Subudhi, S.; Rapin, N.; Misra, V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 2019, 11, 192, doi:10.3390/v11020192.

4. Irving, A.T.; Ahn, M.; Goh, G.; Anderson, D.E.; Wang, L.-F. Lessons from the Host Defences of Bats, a Unique Viral Reservoir. Nature 2021, 589, 363–370, doi:10.1038/s41586-020-03128-0.

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