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The nonvirulent infectious salmon anaemia virus (ISAV-HPR0) is the putative progenitor for virulent-ISAV, and a potential risk factor for the development of infectious salmon anaemia (ISA). Understanding the transmission dynamics of ISAV-HPR0 is fundamental to proper management and mitigation strategies. Here, we demonstrate that ISAV-HPR0 causes prevalent and transient infections in all three production stages of Atlantic salmon in the Faroe Islands. Phylogenetic analysis of the haemagglutinin-esterase gene from 247 salmon showed a clear geographical structuring into two significantly distinct HPR0-subgroups, which were designated G2 and G4. Whereas G2 and G4 co-circulated in marine farms, Faroese broodfish were predominantly infected by G2, and smolt were predominantly infected by G4. This infection pattern was confirmed by our G2- and G4-specific RT-qPCR assays. Moreover, the HPR0 variants detected in Icelandic and Norwegian broodfish were never detected in the Faroe Islands, despite the extensive import of ova from both countries. Accordingly, the vertical transmission of HPR0 from broodfish to progeny is uncommon. Phylogenetic and statistical analysis suggest that HPR0 persists in the smolt farms as “house-strains”, and that new HPR0 variants are occasionally introduced from the marine environment, probably by HPR0-contaminated sea-spray. Thus, high biosecurity—including water and air intake—is required to avoid the introduction of pathogens to the smolt farms.

Viral disease poses a major barrier to sustainable aquaculture, with outbreaks causing large economic losses and growing concerns for fish welfare. Genomic epidemiology can support disease control by providing rapid inferences on viral evolution and disease transmission. In this study, genomic epidemiology was used to investigate salmonid alphavirus (SAV), the causative agent of pancreas disease (PD) in Atlantic salmon. Our aim was to reconstruct SAV subtype-2 (SAV2) diversity and transmission dynamics in recent Norwegian aquaculture, including the origin of SAV2 in regions where this subtype is not tolerated under current legislation. Using nanopore sequencing, we captured ~90% of the SAV2 genome for n = 68 field isolates from 10 aquaculture production regions sampled between 2018 and 2020. Using time-calibrated phylogenetics, we infer that, following its introduction to Norway around 2010, SAV2 split into two clades (SAV2a and 2b) around 2013. While co-present at the same sites near the boundary of Møre og Romsdal and Trøndelag, SAV2a and 2b were generally detected in non-overlapping locations at more Southern and Northern latitudes, respectively. We provide evidence for recent SAV2 transmission over large distances, revealing a strong connection between Møre og Romsdal and SAV2 detected in 2019/20 in Rogaland. We also demonstrate separate introductions of SAV2a and 2b outside the SAV2 zone in Sognefjorden (Vestland), connected to samples from Møre og Romsdal and Trøndelag, respectively, and a likely 100 km Northward transmission of SAV2b within Trøndelag. Finally, we recovered genomes of SAV2a and SAV3 co-infecting single fish in Rogaland, involving novel SAV3 lineages that diverged from previously characterized strains >25 years ago. Overall, this study demonstrates useful applications of genomic epidemiology for tracking viral disease spread in aquaculture.

Structural variants (SVs) are a major source of genetic and phenotypic variation, but remain challenging to accurately type and are hence poorly characterized in most species. We present an approach for reliable SV discovery in non-model species using whole genome sequencing and report 15,483 high-confidence SVs in 492 Atlantic salmon (Salmo salar L.) sampled from a broad phylogeographic distribution. These SVs recover population genetic structure with high resolution, include an active DNA transposon, widely affect functional features, and overlap more duplicated genes retained from an ancestral salmonid autotetraploidization event than expected. Changes in SV allele frequency between wild and farmed fish indicate polygenic selection on behavioural traits during domestication, targeting brain-expressed synaptic networks linked to neurological disorders in humans. This study offers novel insights into the role of SVs in genome evolution and the genetic architecture of domestication traits, along with resources supporting reliable SV discovery in non-model species.

Analysis of pathogen genome variation is essential for informing disease management and control measures in farmed animals. For farmed fish, the standard approach is to use PCR and Sanger sequencing to study partial regions of pathogen genomes, with second and third-generation sequencing tools yet to be widely applied. Here we demonstrate rapid and accurate sequencing of two disease-causing viruses affecting global salmonid aquaculture, salmonid alphavirus (SAV) and infectious salmon anaemia virus (ISAV), using third-generation nanopore sequencing on the MinION platform (Oxford Nanopore Technologies). Our approach complements PCR from infected material with MinION sequencing to recover genomic information that matches near perfectly to Sanger-verified references. We use this method to present the first SAV subtype-6 genome, which branches as the sister to all other SAV lineages in a genome-wide phylogenetic reconstruction. MinION sequencing offers an effective strategy for fast, genome-wide analysis of fish viruses, with major potential applications for diagnostics and robust investigations into the origins and spread of disease outbreaks.

Alzheimer's disease (AD) and other neurodegenerative diseases (NDs) cause substantial health-related and economic burdens, but progress towards preventative or ameliorative treatments has been limited. Genome-wide association studies have identified hundreds of risk loci containing single nucleotide polymorphisms (SNPs) that alter risk for these diseases, but >90% of these SNPs are in noncoding regions, which are cell type-specific and difficult to study. To address this gap, we have characterized the epigenomes of iPSC-derived neuronal and glial cells and performed CRISPRi single cell screening to dissect the molecular and cellular mechanisms underlying 10 ND risk loci. We generated neural progenitors, excitatory neurons, astrocytes and microglia from male and female iPSCs and performed RNA-seq, H3K27ac ChIP-seq, and Promoter Capture Hi-C. We compared these data to those from primary uncultured (ex vivo) human neurons, astrocytes and microglia to compare their enhancer landscapes and ND risk SNP enrichment patterns. To perform CRISPRi single cell screening we integrated a dCas9-KRAB transgene into an iPSC safe harbor locus and developed a novel lentiviral method that allows for efficient and well-tolerated delivery of sgRNAs to iPSC-derived microglia-like cells (iMGLs). iMGLs displayed the strongest correlation of their enhancer landscapes with those of their ex vivo counterparts, as well as highly consistent enrichment of AD and multiple sclerosis (MS) risk SNPs in microglia-specific enhancers. Enhancer/promoter interactions in iMGLs also overlapped significantly with those from ex vivo microglia. As these results suggest that iMGLs are a suitable model for studying ND risk loci, we performed a CRISPRi single cell screen to identify the target genes and pathways affected at 10 AD and MS risk loci. While the enhancer landscapes of iPSC-derived neuronal and glial cells vary in similarity to their ex vivo counterparts, they display similar ND risk SNP enrichments in cell type-specific enhancers, suggesting that disease risk SNP mechanisms are largely recapitulated in iPSC-derived cells. The iMGL epigenome is notably similar to ex vivo microglia, and CRISPRi screening of AD and MS risk loci in these cells will advance our understanding of these diseases and nominate potential therapeutic targets.

Alzheimer's (AD) and other neurological diseases (NDs) cause substantial health-related and economic burdens, but progress toward preventative or ameliorative treatments has been limited. Genome-wide association studies (GWAS) have identified hundreds of risk loci containing single nucleotide polymorphisms (SNPs) that alter ND risk, but >90% of these SNPs are in noncoding regions, which are cell type-specific and difficult to study. To address this limitation, we have combined iPSC-based cell models and functional genomics to pinpoint the causal genes at 10 loci associated with risk for AD and multiple sclerosis (MS). We generated neural progenitors, excitatory neurons, astrocytes and microglia from iPSCs and performed RNA-seq, H3K27ac ChIP-seq and Promoter Capture Hi-C. We compared these data to those from uncultured primary (ex vivo) human brain cells to assess their physiological relevance, and determined global patterns of ND risk SNP enrichments in cell type-specific enhancers. We transduced iPSC-derived microglia-like cells (iMGLs) with a lentiviral sgRNA library targeting AD and MS risk SNPs and performed scRNA-seq to identify altered genes and cellular pathways. iPSC-derived cell types displayed gene signatures and enhancer patterns consistent with their cell identities, with iMGLs displaying the strongest similarity to primary cells. Neuronal and glial cells from both iPSC and primary brain sources show consistent enrichment of ND risk SNPs in cell type-specific enhancers, with microglia showing enrichment of AD and MS SNPs. Enhancer/promoter interactions identified in iMGLs and primary microglia nominated potential target genes of these SNPs, and these predictions were tested by CRISPRi single cell screening. We identified multiple novel AD and MS risk genes, including the transcription factor MAF, which is regulated by both AD and MS risk SNPs, and ELMO1, which we characterize as a novel MS risk gene involved in microglial homeostasis. The combination of iPSC-based cell models and functional genomics allowed for systematic perturbation of AD and MS risk SNPs, revealing previously uncharacterized risk genes linking neuroimmune pathways to disease pathophysiology. Ongoing studies are exploring the role of altered expression levels of MAF, ELMO1 and other screen hits in microglial phenotypes, including effects on neurons and astrocytes in co-culture and organoid systems.

Objective Parkinson's disease (PD) presents clinically with several motor subtypes that exhibit variable treatment response and prognosis. Here, we investigated genetic variants for their potential association with PD motor phenotype and progression. Methods We screened 10 SNPs, previously associated with PD risk, for association with tremor‐dominant (TD) versus postural‐instability gait disorder (PIGD) motor subtypes. SNPs that correlated with the TD/PIGD ratio in a discovery cohort of 251 PD patients were then evaluated in a multi‐site replication cohort of 559 PD patients. SNPs associated with motor phenotype in both cross‐sectional cohorts were next evaluated for association with (1) rates of motor progression in a longitudinal subgroup of 230 PD patients and (2) brain alpha‐synuclein (SNCA) expression in the GTEx (Genotype‐Tissue Expression project) consortium database. Results Genotype at rs356182, near SNCA, correlated with the TD/PIGD ratio in both the discovery (Bonferroni‐corrected P = 0.04) and replication cohorts (P = 0.02). The rs356182 GG genotype was associated with a more tremor‐predominant phenotype and predicted a slower rate of motor progression (1‐point difference in annual rate of UPDRS‐III motor score change, P = 0.01). The rs356182 genotype was associated with SNCA expression in the cerebellum (P = 0.005). Interpretation Our study demonstrates that the GG genotype at rs356182 provides molecular definition for a clinically important endophenotype associated with (1) more tremor‐predominant motor phenomenology, (2) slower rates of motor progression, and (3) decreased brain expression of SNCA. Such molecularly defined endophenotyping in PD may benefit both clinical trial design and tailoring of clinical care as we enter the era of precision medicine.

Background Alzheimer’s disease (AD) and other neurodegenerative diseases (NDs) cause substantial health‐related and economic burdens, but progress towards preventative or ameliorative treatments has been limited. Genome‐wide association studies have identified hundreds of risk loci containing single nucleotide polymorphisms (SNPs) that alter risk for these diseases, but >90% of these SNPs are in noncoding regions, which are cell type‐specific and difficult to study. To address this gap, we have characterized the epigenomes of iPSC‐derived neuronal and glial cells and performed CRISPRi single cell screening to dissect the molecular and cellular mechanisms underlying 10 ND risk loci. Method We generated neural progenitors, excitatory neurons, astrocytes and microglia from male and female iPSCs and performed RNA‐seq, H3K27ac ChIP‐seq, and Promoter Capture Hi‐C. We compared these data to those from primary uncultured (ex vivo) human neurons, astrocytes and microglia to compare their enhancer landscapes and ND risk SNP enrichment patterns. To perform CRISPRi single cell screening we integrated a dCas9‐KRAB transgene into an iPSC safe harbor locus and developed a novel lentiviral method that allows for efficient and well‐tolerated delivery of sgRNAs to iPSC‐derived microglia‐like cells (iMGLs). Result iMGLs displayed the strongest correlation of their enhancer landscapes with those of their ex vivo counterparts, as well as highly consistent enrichment of AD and multiple sclerosis (MS) risk SNPs in microglia‐specific enhancers. Enhancer/promoter interactions in iMGLs also overlapped significantly with those from ex vivo microglia. As these results suggest that iMGLs are a suitable model for studying ND risk loci, we performed a CRISPRi single cell screen to identify the target genes and pathways affected at 10 AD and MS risk loci. Conclusion While the enhancer landscapes of iPSC‐derived neuronal and glial cells vary in similarity to their ex vivo counterparts, they display similar ND risk SNP enrichments in cell type‐specific enhancers, suggesting that disease risk SNP mechanisms are largely recapitulated in iPSC‐derived cells. The iMGL epigenome is notably similar to ex vivo microglia, and CRISPRi screening of AD and MS risk loci in these cells will advance our understanding of these diseases and nominate potential therapeutic targets.

The ability to control transgene expression both temporally and quantitatively in human-relevant cells and tissues is a cornerstone of biomedical research. Additionally, precise transgene control is crucial for optimizing human cell-based gene therapies. Human pluripotent stem cells (hPSCs) have facilitated major advances in disease modeling and the potential for regenerative medicine. Still, they are significantly limited by the lack of inducible transgenic systems that avoid silencing but maintain robust inducibility after differentiation to defined cell lineages. Here we systematically characterize the leakiness, inducibility, and tunability of multiple existing and novel transgenic systems in hPSCs and differentiated macrophages and microglia. Notably, we report the application of a small molecule-mediated splicing switch (X ) that allows for tunable transgene expression both before and after differentiation, without the large protein tags required for current state-of-the-art degron-based methods. We use X to achieve tight control of reporter genes, overexpression of multiple neurodegeneration-associated genes, and Cas9-mediated genome editing. We also characterize the current limitations of this system and describe approaches that can alleviate some of these limitations. By assessing multiple transgenic systems whose inducibility spans the transcriptional, post-transcriptional, and post-translational levels, we highlight and improve upon a major technical challenge that hinders basic, translational, and clinical research in physiological human-based systems.

Background Alzheimer's (AD) and other neurological diseases (NDs) cause substantial health‐related and economic burdens, but progress toward preventative or ameliorative treatments has been limited. Genome‐wide association studies (GWAS) have identified hundreds of risk loci containing single nucleotide polymorphisms (SNPs) that alter ND risk, but >90% of these SNPs are in noncoding regions, which are cell type‐specific and difficult to study. To address this limitation, we have combined iPSC‐based cell models and functional genomics to pinpoint the causal genes at 10 loci associated with risk for AD and multiple sclerosis (MS). Methods We generated neural progenitors, excitatory neurons, astrocytes and microglia from iPSCs and performed RNA‐seq, H3K27ac ChIP‐seq and Promoter Capture Hi‐C. We compared these data to those from uncultured primary (ex vivo) human brain cells to assess their physiological relevance, and determined global patterns of ND risk SNP enrichments in cell type‐specific enhancers. We transduced iPSC‐derived microglia‐like cells (iMGLs) with a lentiviral sgRNA library targeting AD and MS risk SNPs and performed scRNA‐seq to identify altered genes and cellular pathways. Results iPSC‐derived cell types displayed gene signatures and enhancer patterns consistent with their cell identities, with iMGLs displaying the strongest similarity to primary cells. Neuronal and glial cells from both iPSC and primary brain sources show consistent enrichment of ND risk SNPs in cell type‐specific enhancers, with microglia showing enrichment of AD and MS SNPs. Enhancer/promoter interactions identified in iMGLs and primary microglia nominated potential target genes of these SNPs, and these predictions were tested by CRISPRi single cell screening. We identified multiple novel AD and MS risk genes, including the transcription factor MAF, which is regulated by both AD and MS risk SNPs, and ELMO1, which we characterize as a novel MS risk gene involved in microglial homeostasis. Conclusions The combination of iPSC‐based cell models and functional genomics allowed for systematic perturbation of AD and MS risk SNPs, revealing previously uncharacterized risk genes linking neuroimmune pathways to disease pathophysiology. Ongoing studies are exploring the role of altered expression levels of MAF, ELMO1 and other screen hits in microglial phenotypes, including effects on neurons and astrocytes in co‐culture and organoid systems.