Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus

Infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) are economically important pathogens of the salmonid aquaculture industry. Atlantic salmon were challenged by intraperitoneal injection (i.p.) with either virus followed by time-course sampling. Cohabiting fish in the IPNV challenge were also sampled. Kidney tissue was analysed using a TaqMan real-time PCR assay to measure the expression of a range of host immune genes in relation to the endogenous control, elongation factor 1 alpha (ELF). Host genes measured included Mx, type I and type II interferon (IFN), gammaIFN induced protein (gammaIP), interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Viral levels were also measured. In i.p. injected fish, both viruses greatly induced expression of Mx, gammaIP, type I and type II IFN by day 6 post-infection, however only ISAV caused substantial mortality. Some differences between the expression kinetics produced by both viruses were noted. Infection with ISAV increased IL-1beta expression following day 6, but no effect was seen in fish infected with IPNV. Neither virus induced TNF-alpha expression. This study confirms the presence of both type I and type II IFN responses and their induced genes in Atlantic salmon upon infection with an orthomyxovirus and a birnavirus.     

Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.

Infectious pancreatic necrosis (IPN) virus (IPNV) infection in Atlantic salmon Salmo salar L. post-smolts and its influence on the outcome of secondary infections with infectious salmon anaemia (ISA) virus (ISAV) or Vibrio salmonicida were studied. The infections with ISAV or V salmonicida were performed both in a period of acute IPN and in the following IPNV carrier stage, 3 and 6 to 8 wk after experimental IPNV challenge, respectively. An IPNV carrier condition at low virus titre did not influence the mortality rates after secondary infections. Neither the ISAV infection nor the V. salmonicida infection in experimentally induced IPNV carriers resulted in mortalities different from those observed after challenge of IPNV-free fish. At higher IPNV titres in Atlantic salmon with acute IPN, the outcome of secondary infections was quite different from that observed in IPNV-free fish and in IPNV carriers. In 2 different experiments significantly more fish died when fish with acute IPN were infected with V salmonicida than when fish were infected with V salmonicida alone. Mortality also started earlier in the double-infected group than in the group challenged with V. salmonicida alone, 3 to 4 and 8 d after V salmonicida infection, respectively. Similar results were observed independent of whether mortalities due to IPN alone were registered in the experiments. When Atlantic salmon with acute IPN were infected with ISAV, significantly fewer fish died than when fish were infected with ISAV alone. The ongoing IPNV infection seemed to provide some protection against development of ISA.     

Rapid detection of viral haemorrhagic septicaemia virus in fish by ELISA

An ELISA for rapid detection of viral haemorrhagic septicaemia virus (VHSV, Egtved virus) in fish tissue was developed and tested with regard to sensitivity. The test is a valuable addition to traditional isolation of VHSV in cell culture when a rapid diagnosis is needed.     

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.     

Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV)

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.     

Sea trout as a carrier of infectious salmon anaemia virus

Sea trout did not develop symptoms or gross clinical signs of infectious salmon anaemia (ISA) when injected with ascites from salmon, but ISA virus was propagated and haematocrit values dropped. The virus was transmitted from trout to salmon.     

Comparative susceptibility of turbot Scophthalmus maximus to different genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) disease has exerted a significant impact on the development of turbot aquaculture in the British Isles. The source of such outbreaks is believed to be naturally occurring marine isolates of viral haemorrhagic septicaemia virus (VHSV), which are endemic in the marine environment of Northern Europe. Genetic studies have classified these marine VHSV isolates into genotypes based on their geographic rather than host-species origin. This study set out to explore the hypothesis that susceptibility of turbot to VHSV might be genotype specific. Immersion infection of turbot with a range of isolates, selected according to genotype, identified significant differences between susceptibility and genotype. Viruses belonging to Genotypes Ib (Baltic marine isolates) and III (North Sea/E. Atlantic marine isolates) caused significantly higher mortality than isolates from Genotypes Ia (isolates associated with rainbow trout aquaculture) and II (Baltic marine isolates). This study serves to highlight the importance of thoroughly investigating the susceptibility of any given species to the range of pathogens to which they might be exposed prior to considering them resistant to any disease. Furthermore, it highlights different risk factors that might be associated with turbot aquaculture undertaken in different environments. Finally, an increased knowledge of the relative virulence of different isolates in turbot will assist in understanding virulence determinants, which could lead to advances in disease control.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.     

Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

The resistance of rainbow trout (Oncorhynchus mykiss) to an infectious haematopoietic necrosis virus (IHNV) challenge following a preceding non-lethal infection with infectious pancreatic necrosis virus (IPNV) was investigated through experimental dual infections. Trout initially infected with IPNV were inoculated 14 days later with IHNV. Single infections of trout with 1 of the 2 viruses or with cell culture supernatant were also carried out and constituted control groups. No mortality was noted in fish after a single infection with IPNV. This virus had no influence on the head kidney leucocyte phagocytic activity and plasma haemolytic complement activity. IHNV induced a high mortality (72%) and reduced the macrophage phagocytic activity and complement haemolytic activity. It also induced a late production of anti-IHNV antibodies which occurred after clearance of the virus in the fish. In trout co-infected with both viruses, a mortality rate of 2% occurred and the immune parameters were similar to those observed in the fish infected with IPNV only, demonstrating that in co-infected trout IPNV inhibits the effects of IHNV. The studied parameters did not allow us to define the mechanism of interference occurring between these 2 viruses, but some hypothesis are put forward to explain the interference between the 2 viruses.     

Prevalence of viral haemorrhagic septicaemia virus in Danish marine fishes and its occurrence in new host species

In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine waters around Denmark, staff from the Danish Institute for Food and Veterinary Research participated in 5 research cruises during 1998 to 2002 as a follow-up to 4 research cruises performed in 1996 to 1997. In total, 16,655 fish were examined virologically as 3569 samples. Forty fish species and 3 invertebrate species were represented. VHSV was isolated from 133 samples representing 8 species: herring Clupea harengus, sprat Sprattus sprattus, dab Limanda limanda, flounder Platichthys flesus, plaice Pleuronectes platessa, cod Gadus morhua, sand eel Ammodytes sp. and sand goby Pomatochistus minutus. Calculations showed that VHSV was more prevalent in the Baltic Sea in an area between Zealand and the island of Bornholm and the waters surrounding Bornholm than in the Kattegat, Skagerrak and along the North Sea coast of Denmark. This is the first report on the isolation of VHSV from dab, flounder and plaice and the first publication on VHSV from sand eel from Europe and sand goby.     

Genetic analysis of infectious salmon anaemia virus (ISAV) from Scotland

The nucleotide sequences of segments 2 and 8 of infectious salmon anaemia virus (ISAV) isolates from Scotland were determined. These were used to predict amino acid sequences of the products of these segments. The sequences were compared with those previously published for ISAV from Norway and North America. Nucleotide and amino acid variations were found in the Scottish isolate, indicating that it is not identical to those Norwegian or North American strains. Although phylogenetic analysis of the sequences was not possible, it was clear that the Scottish isolate is more closely related to the Norwegian strain than the North American. Sequencing further isolates that are geographically or temporally separated will be important in advancing understanding of the epidemiology of this important disease.     

Experimental susceptibility of turbot Scophthalmus maximus to viral haemorrhagic septicaemia virus isolated from cultivated turbot

Juvenile pathogen-free turbot were infected with a viral haemorrhagic septicaemia virus (VHSV) isolate recovered from turbot cultivated on the island of Gigha, West Scotland. Mortality of 100% was recorded in fish infected via the intra-peritoneal (i.p.) route. Horizontal transmission of VHSV in sea water was demonstrated by cohabitation of naive fish with i.p. infected fish at a ratio of 1:1. The total cumulative average mortality in cohabiting fish was 60% by 60 d post-infection. Turbot infected via an immersion route exhibited a cumulative average mortality of 71% by the end of the experiment. VHSV identified by enzyme-linked immunosorbent assay (ELISA) was recovered from both organ (kidney and spleen) and brain samples of individual fish that died following infection by all experimental routes. These findings pose significant implications regarding the persistence of VHSV and its role in limiting natural populations of marine fish species. In addition, the establishment of infection models for the transmission of VHSV in sea water is of fundamental importance to the development of anti-VHSV vaccines in important commercial species such as turbot.     

Detection of infectious salmon anaemia virus (ISAV) by in situ hybridisation

An in situ hybridisation method was developed to detect infectious salmon anaemia virus (ISAV) in fixed tissues from Atlantic salmon Salmo salar L. Three DNA probes detected ISAV in heart, liver, kidney, spleen, caeca, and mid-gut from infected farmed Atlantic salmon obtained from a natural outbreak of ISA. The strongest signals were obtained using Probe S8, from Segment 8 of ISAV. Hybridisation was most prominent in the endothelial cells of heart tissue. The probes reacted specifically with ISAV; no hybridisation was evident in uninfected tissues from Atlantic salmon. Importantly, the probes did not cross react with the pathogens IHNV (haematopoietic necrosis virus), IPNV (infectious pancreatic necrosis virus), SPDV (salmon pancreas disease virus) and VHSV (viral haemorrhagic septicemia virus).