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.

No Evidence of the Vertical Transmission of Non-Virulent Infectious Salmon Anaemia Virus (ISAV-HPR0) in Farmed Atlantic Salmon

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.

Protection of Teleost Fish against Infectious Diseases through Oral Administration of Vaccines: Update 2021

Immersion and intraperitoneal injection are the two most common methods used for the vaccination of fish. Because both methods require that fish are handled and thereby stressed, oral administration of vaccines as feed supplements is desirable. In addition, in terms of revaccination (boosting) of adult fish held in net pens, oral administration of vaccines is probably the only feasible method to obtain proper protection against diseases over long periods of time. Oral vaccination is considered a suitable method for mass immunization of large and stress-sensitive fish populations. Moreover, oral vaccines may preferably induce mucosal immunity, which is especially important to fish. Experimental oral vaccine formulations include both non-encapsulated and encapsulated antigens, viruses and bacteria. To develop an effective oral vaccine, the desired antigens must be protected against the harsh environments in the stomach and gut so they can remain intact when they reach the lower gut/intestine where they normally are absorbed and transported to immune cells. The most commonly used encapsulation method is the use of alginate microspheres that can effectively deliver vaccines to the intestine without degradation. Other encapsulation methods include chitosan encapsulation, poly D,L-lactide-co-glycolic acid and liposome encapsulation. Only a few commercial oral vaccines are available on the market, including those against infectious pancreatic necrosis virus (IPNV), Spring viremia carp virus (SVCV), infectious salmon anaemia virus (ISAV) and Piscirickettsia salmonis. This review highlights recent developments of oral vaccination in teleost fish.

Short communication: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anaemia virus in seawater

Infectious salmon anaemia virus (ISAV) is the cause of an important waterborne disease of farmed Atlantic salmon. Detection of virus in water samples may constitute an alternative method to sacrificing fish for surveillance of fish populations for the presence of ISA-virus. We aimed to evaluate different membrane filters and buffers for concentration and recovery of ISAV in seawater, prior to molecular detection. One litre each of artificial and natural seawater was spiked with ISAV, followed by concentration with different filters and subsequent elution with different buffers. The negatively charged MF hydrophilic membrane filter, combined with NucliSENS® lysis buffer, presented the highest ISAV recovery percentages with 12.5 ± 1.3% by RT-qPCR and 31.7 ± 10.7% by RT-ddPCR. For the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter, combined with NucliSENS® lysis buffer, the ISAV recovery percentages were 3.4 ± 0.1% by RT-qPCR and 10.8 ± 14.2% by RT-ddPCR. The limits of quantification (LOQ) were estimated to be 2.2 x 103 ISAV copies/L of natural seawater for both RT-qPCR and RT-ddPCR. The ISAV concentration method was more efficient in natural seawater.

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

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.

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

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.