Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing

Background: Infectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that currently represents a large problem for salmonid aquaculture worldwide. Prevention and treatment methods are only partially effective. Genetic selection and genome engineering strategies have potential to develop ISAV resistant salmon stocks. However, this requires a detailed understanding of the genomic regulation of ISAV pathogenesis. Here, we used single cell RNA sequencing on a salmonid cell line to provide a high dimensional insight into the transcriptional landscape that underpin host-virus interactions during ISAV infection at the single cell level. Results: Salmon head kidney 1 (SHK-1) cells were single-cell RNA sequenced before challenge, and at 24h, 48h, and 96h post-ISAV challenge. The results revealed marked changes in the host transcriptome at 48h and 96h post-infection, even in uninfected cells, potentially suggesting paracrine signalling. This paracrine activation of uninfected cells seemed to be unspecific, involving pathways such as mRNA sensing, ubiquitination or proteasome, and also the up-regulation of the mitochondrial ribosome genes. At 24h post infection, cells showed expression signatures consistent with viral entry, with up-regulation of genes such as PI3K, FAK or JNK. At 48h and 96h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Conclusions: This study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection, and revealed potential host-virus interactions at the cellular level. The results highlight the value of single-cell sequencing to characterise cell culture models of viral infection, and the results can be exploited in future functional studies to increase the resistance of Atlantic salmon to ISAV.

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Multiplication and haemadsorbing activity of infectious salmon anaemia virus in the established Atlantic salmon cell line.

Journal of General Virology ◽

10.1099/0022-1317-78-8-1891 ◽

1997 ◽

Vol 78 (8) ◽

pp. 1891-1895 ◽

Cited By ~ 15

Author(s):

A I Sommer ◽

S Mennen

Keyword(s):

Atlantic Salmon ◽

Cell Line ◽

Infectious Salmon Anaemia Virus ◽

Infectious Salmon Anaemia

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DOP23 Single-cell RNA sequencing identifies an important role for class I histone-deacetylase enzymes in intestinal myofibroblasts from patients with Crohn’s Disease strictures

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab073.062 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S062-S062

Author(s):

A Lewis ◽

B Pan-Castillo ◽

G Berti ◽

C Felice ◽

H Gordon ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Line ◽

Rna Sequencing ◽

Histone Deacetylase ◽

Chromatin Accessibility ◽

Collagen I ◽

Class I ◽

Rna Seq ◽

Single Cell Rna Sequencing

Abstract Background Histone-deacetylase (HDAC) enzymes are a broad class of ubiquitously expressed enzymes that modulate histone acetylation, chromatin accessibility and gene expression. In models of Inflammatory bowel disease (IBD), HDAC inhibitors, such as Valproic acid (VPA) are proven anti-inflammatory agents and evidence suggests that they also inhibit fibrosis in non-intestinal organs. However, the role of HDAC enzymes in stricturing Crohn’s disease (CD) has not been characterised; this is key to understanding the molecular mechanism and developing novel therapies. Methods To evaluate HDAC expression in the intestine of SCD patients, we performed unbiased single-cell RNA sequencing (sc-RNA-seq) of over 10,000 cells isolated from full-thickness surgical resection specimens of non-SCD (NSCD; n=2) and SCD intestine (n=3). Approximately, 1000 fibroblasts were identified for further analysis, including a distinct cluster of myofibroblasts. Changes in gene expression were compared between myofibroblasts and other resident intestinal fibroblasts using the sc-RNA-seq analysis pipeline in Partek. Changes in HDAC expression and markers of HDAC activity (H3K27ac) were confirmed by immunohistochemistry in FFPE tissue from patient matched NSCD and SCD intestine (n=14 pairs). The function of HDACs in intestinal fibroblasts in the CCD-18co cell line and primary CD myofibroblast cultures (n=16 cultures) was assessed using VPA, a class I HDAC inhibitor. Cells were analysed using a variety of molecular techniques including ATAC-seq, gene expression arrays, qPCR, western blot and immunofluorescent protein analysis. Results Class I HDAC (HDAC1, p= 2.11E-11; HDAC2, p= 4.28E-11; HDAC3, p= 1.60E-07; and HDAC8, p= 2.67E-03) expression was increased in myofibroblasts compared to other intestinal fibroblasts subtypes. IHC also showed an increase in the percentage of stromal HDAC2 positive cells, coupled with a decrease in the percentage of H3K27ac positive cells, in the mucosa overlying SCD intestine relative to matched NSCD areas. In the CCD-18co cell line and primary myofibroblast cultures, VPA reduced chromatin accessibility at Collagen-I gene promoters and suppressed their transcription. VPA also inhibited TGFB-induced up-regulation of Collagen-I, in part by inhibiting TGFB1|1/SMAD4 signalling. TGFB1|1 was identified as a mesenchymal specific target of VPA and siRNA knockdown of TGFB1|1 was sufficient suppress TGFB-induced up-regulation of Collagen-I. Conclusion In SCD patients, class I HDAC expression is increased in myofibroblasts. Class I HDACs inhibitors impair TGFB-signalling and inhibit Collagen-I expression. Selective targeting of TGFB1|1 offers the opportunity to increase treatment specificity by selectively targeting meschenymal cells.

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Exploring genetic resistance to Infectious Salmon Anaemia Virus in Atlantic salmon by genome-wide association and RNA sequencing

10.1101/2020.09.08.287052 ◽

2020 ◽

Author(s):

O. Gervais ◽

A. Barria ◽

A. Papadopoulou ◽

R. Gratacap ◽

B. Hillestad ◽

...

Keyword(s):

Atlantic Salmon ◽

Rna Sequencing ◽

Post Infection ◽

Snp Array ◽

Genome Wide Association ◽

Control Fish ◽

Infectious Salmon Anaemia Virus ◽

Transcriptomic Response ◽

Genome Wide ◽

A Genome

ABSTRACTInfectious Salmonid Anaemia Virus (ISAV) causes a notifiable disease that poses a large threat for Atlantic salmon breeders and producers worldwide. There is no fully effective treatment or vaccine, and therefore selective breeding to increase resistance to ISAV in commercial strains of Atlantic salmon is a promising avenue for disease prevention. Genomic selection and potentially genome editing can be applied to enhance host resistance, and these approaches benefit from improved knowledge of the genetic and functional basis of the target trait. The aim of this study was to characterise the genetic architecture of resistance to ISAV in a commercial Atlantic salmon population and study its underlying functional genomic basis using RNA Sequencing. A total of 2,833 Atlantic salmon parr belonging to 194 families were exposed to ISAV in a cohabitation challenge in which cumulative mortality reached 63% over 55 days. A total of 1,353 animals were genotyped using a 55K SNP array, and the estimate of heritability for the trait of binary survival was 0.33 (±0.04). A genome-wide association analysis confirmed that resistance to ISAV was a polygenic trait, albeit a genomic region in chromosome 13 was significantly associated with resistance and explained 3% of the genetic variance. RNA sequencing of the heart of 16 infected (7 and 14 days post infection) and 8 control fish highlighted 4,927 and 2,437 differentially expressed genes at 7 and 14 days post infection respectively. The complement and coagulation pathway was down-regulated, while several metabolic pathways were up-regulated in infected fish compared to controls. The interferon pathway was mildly activated at 7 days and showed no sign of up-regulation at 14 days post infection, implying a crosstalk between host and virus. Comparison of the transcriptomic response of fish with high and low breeding values for resistance (4 high resistance and 4 low resistance animals per time point) highlighted TRIM25 as being up-regulated in resistant fish, suggesting it may be a key antiviral gene involved in the functional genetic basis of resistance to ISAV.

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Infectious salmon anaemia virus (ISAV) isolates induce distinct gene expression responses in the Atlantic salmon (Salmo salar) macrophage/dendritic-like cell line TO, assessed using genomic techniques

Molecular Immunology ◽

10.1016/j.molimm.2009.06.015 ◽

2009 ◽

Vol 46 (15) ◽

pp. 2955-2974 ◽

Cited By ~ 49

Author(s):

Samuel T. Workenhe ◽

Tiago S. Hori ◽

Matthew L. Rise ◽

Molly J.T. Kibenge ◽

Frederick S.B. Kibenge

Keyword(s):

Gene Expression ◽

Atlantic Salmon ◽

Cell Line ◽

Salmo Salar ◽

Infectious Salmon Anaemia Virus ◽

Atlantic Salmon Salmo Salar ◽

Distinct Gene ◽

Infectious Salmon Anaemia

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Exploring genetic resistance to infectious salmon anaemia virus in Atlantic salmon by genome-wide association and RNA sequencing

BMC Genomics ◽

10.1186/s12864-021-07671-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

O. Gervais ◽

A. Barria ◽

A. Papadopoulou ◽

R. L. Gratacap ◽

B. Hillestad ◽

...

Keyword(s):

Atlantic Salmon ◽

Rna Sequencing ◽

Post Infection ◽

Genome Wide Association ◽

Control Fish ◽

Infectious Salmon Anaemia Virus ◽

Transcriptomic Response ◽

Breeding Values ◽

Genome Wide ◽

Interferon Pathway

Abstract Background Infectious Salmonid Anaemia Virus (ISAV) causes a notifiable disease that poses a large threat for Atlantic salmon (Salmo salar) aquaculture worldwide. There is no fully effective treatment or vaccine, and therefore selective breeding to increase resistance to ISAV is a promising avenue for disease prevention. Genomic selection and potentially genome editing can be applied to enhance host resistance, and these approaches benefit from improved knowledge of the genetic and functional basis of the target trait. The aim of this study was to characterise the genetic architecture of resistance to ISAV in a commercial Atlantic salmon population and study its underlying functional genomic basis using RNA Sequencing. Results A total of 2833 Atlantic salmon parr belonging to 194 families were exposed to ISAV in a cohabitation challenge in which cumulative mortality reached 63% over 55 days. A total of 1353 animals were genotyped using a 55 K SNP array, and the estimate of heritability for the trait of binary survival was 0.13–0.33 (pedigree-genomic). A genome-wide association analysis confirmed that resistance to ISAV was a polygenic trait, albeit a genomic region in chromosome Ssa13 was significantly associated with resistance and explained 3% of the genetic variance. RNA sequencing of the heart of 16 infected (7 and 14 days post infection) and 8 control fish highlighted 4927 and 2437 differentially expressed genes at 7 and 14 days post infection respectively. The complement and coagulation pathway was down-regulated in infected fish, while several metabolic pathways were up-regulated. The interferon pathway showed little evidence of up-regulation at 7 days post infection but was mildly activated at 14 days, suggesting a potential crosstalk between host and virus. Comparison of the transcriptomic response of fish with high and low breeding values for resistance highlighted TRIM25 as being up-regulated in resistant fish. Conclusions ISAV resistance shows moderate heritability with a polygenic architecture, but a significant QTL was detected on chromosome 13. A mild up-regulation of the interferon pathway characterises the response to the virus in heart samples from this population of Atlantic salmon, and candidate genes showing differential expression between samples with high and low breeding values for resistance were identified.

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