Genomic analysis of the host response to nervous necrosis virus in Atlantic cod (Gadus morhua) brain.

Genome sequencing combined with transcriptome profiling promotes exploration of defence against pathogens and discovery of immune genes. Based on sequences from the recently released genome of Atlantic cod, a genome-wide oligonucleotide microarray (ACIQ-1) was designed and used for analyses of gene expression in the brain during infection with nervous necrosis virus (NNV). A challenge experiment with NNV was performed with Atlantic cod juveniles and brain samples from virus infected and uninfected fish were used for microarray analysis. Expression of virus induced genes increased at 5 days post challenge and persisted at stable level to the last sampling at 25 days post challenge. A large fraction of the up-regulated genes (546 features) were known or expected to have immune functions and most of these have not previously been characterized in Atlantic cod. Transcriptomic changes induced by the virus involved strong activation of genes associated with interferon and tumour necrosis factor related responses and acute inflammation. Up-regulation of genes involved in adaptive immunity suggested a rapid recruitment of B and T lymphocytes to the NNV infected brain. QPCR analyses of 15 candidate genes of innate immunity showed rapid induction by poly(I:C) in Atlantic cod larvae cells suggesting an antiviral role. Earliest and greatest expression changes after poly I:C stimulation was observed for interferon regulatory factors IRF4 and IRF7. Comparative studies between teleost species provided new knowledge about the evolution of innate antiviral immunity in fish. A number of genes is present or responds to viruses only in fish. Innate immunity of Atlantic cod is characterized by selective expansion of several medium-sized multigene families with ribose binding domains. An interesting finding was the high representation of three large gene families among the early antiviral genes, including tripartite motif proteins (TRIM) and proteins with PRY-SPRY and NACHT domains. The latter two with respectively 52 and 114 members in Atlantic cod have gone through expansions in different groups of fish. These proteins most likely have ligand binding properties and their propagation could be linked to the loss of MHC class II in the Atlantic cod genome.

Susceptibility of Atlantic cod Gadus morhua juveniles to different routes of experimental challenge with infectious pancreatic necrosis virus (IPNV).

Atlantic cod Gadus morhua L. juveniles weighing 40 g were challenged with infectious pancreatic necrosis virus (IPNV) by intraperitoneal (i.p.) or intramuscular (i.m.) injection or by bath. The amount of infectious virus was determined over 6 wk in head kidney, heart and pylorus tissues. No mortality or clinical signs were observed in either of the challenged groups. However, 6 wk after challenge virus was still present in the fish, which shows that IPNV can persist asymptomatically in cod. I.p. and i.m. injections were the most efficient routes of challenge giving the highest virus recovery. The prevalence of individuals with a viral titre > or = 500 infectious units g(-1) tissue was lower in the group of fish challenged by bath; thus bath was a less efficient route of challenge than injection. Our data also show that pylorus and head kidney are target organs for IPNV in cod, and levels of virus recovery were not considerably different between these 2 organs. Challenged by injection, the cod heart is also a target organ for IPNV. Compared to head kidney and pylorus, the heart seems to have a minor role in virus multiplication. Virus was also recovered from cohabiting fish, demonstrating that covertly infected cod may represent a reservoir of infectious IPNV for surrounding fish populations. Expression analysis of selected cod immune genes showed that i.p. injection of IPNV induced gene expression of ISG15 and LGP2, markers for the innate antiviral defence, while expression of markers for the inflammatory response (interleukins IL-1 beta, IL-8, IL-10) was not significantly increased.

Susceptibility of spotted wolffish Anarhichas minor to experimental infection with nodavirus and infectious pancreatic necrosis virus.

The spotted wolffish Anarhichas minor is a promising new species for cold-water aquaculture. The broad host-range of piscine nodavirus (NV) and infectious pancreatic necrosis virus (IPNV) makes them potentially pathogenic to new fish species in aquaculture. IPNV and NV strains highly pathogenic in farmed Atlantic salmon Salmo salar and halibut Hippoglossus hippoglassus, respectively, in Norway were used for the challenge of spotted wolffish. In general, water-borne infection with IPNV and NV resulted in significant mortality among juveniles <1 g. Cumulative mortality after bath-challenge and cohabitation was 60 to 75% in the smallest juveniles (0.3 g). Intramuscular and intraperitoneal injection of NV was 100% lethal to wolffish of 10 g, and the groups at 12 degrees C died before those at 7 degrees C. No cohabitants of this size died, but NV was still detectable in these individuals after 10 wk. A persistent IPNV infection with low mortality developed in bath-challenged juveniles of 0.7 g, in which IPNV was still detectable 4 mo later. This study comprises a demonstration of experimental viral infections in cultured spotted wolffish, although to date no natural outbreaks of viral diseases have been reported in this species.

Effect of poly I:C on the expression of Mx proteins and resistance against infection by infectious salmon anaemia virus in Atlantic salmon.

Mx proteins are induced by type I interferons (IFN alpha and beta) in mice and humans and inhibit the replication of orthomyxoviruses and some other single-stranded RNA viruses. Recently, Mx genes have been cloned from Atlantic salmon. Mx transcripts were shown to be induced in head-kidney, liver and gills of the fish by the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C). In the present work we have studied expression of Mx protein in organs of Atlantic salmon treated with poly I:C. A quantitative immunoblot method was established to monitor expression of Mx protein and to compare relative amounts of Mx protein in different organs. Treatment of Atlantic salmon with poly I:C increased the relative amount of Mx protein in liver, stomach, hindgut, head-kidney and spleen. In gills the levels of Mx protein were similar in control fish and poly I:C treated fish. Immunohistochemistry of tissue sections from liver, head-kidney and gills from poly I:C treated fish was in accordance with the immunoblotting data and showed staining for Mx protein in several different cell types. Classification of infectious salmon anaemia virus as an orthomyxovirus makes it a putative target for Atlantic salmon Mx protein. Atlantic salmon treated with poly I:C showed reduced cumulative mortality compared to the control fish when challenged with infectious salmon anaemia virus (ISAV) by intraperitoneal injection. This demonstrates that poly I:C has some protective effect against ISAV in vivo.