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The article focuses on a study conducted to investigate the outbreak of viral nervous necrosis (VNN) in the farmed Atlantic cod fishes in Norway during 2006. It discusses the genetic factors causing VNN in farmed juvenile and adult marine fish species, and gives an overview of the investigations done by the study on the infected fishes. Also discussed are the disease symptoms found in fishes during the study, and explores its findings on the pathogenesis and etiology of VNN.

The article presents a study which examines a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that affected both a neurological and septicaemic nature in seawater-farmed rainbow trout, Oncorhynchus mykiss, in Storfjorden, Norway. VHS refers to a severe condition which affects farmed seawater-farmed rainbow trout in Europe. For the study, researchers gathered rainbow trout tissue samples and submitted them to the National Veterinary Institute (NVI). Pathological results including internal haemorrhages and necrosis of haematopoietic showed consistency with the haemorrhagic type of VHS.

The present paper describes, for the first time, clinical signs and pathological findings of pancreas disease (PD) in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea water in Norway. Similarities and differences with reports of PD from Ireland and Scotland are discussed. Samples of 68 rainbow trout from disease outbreaks on 14 farms and from 155 Atlantic salmon from outbreaks on 20 farms collected from 1996 to 2004 were included in the present study. The histopathological findings of PD in Atlantic salmon and rainbow trout in sea water were similar. Acute PD, characterized by acute necrosis of exocrine pancreatic tissues, was detected in nine Atlantic salmon and three rainbow trout. Salmonid alphavirus (SAV) was identified in acute pancreatic necroses by immunohistochemistry. Most fish showed severe loss of exocrine pancreatic tissue combined with chronic myositis. Myocarditis was often but not consistently found. Kidneys from 40% and 64% of the rainbow trout and Atlantic salmon, respectively, had cells along the sinusoids that were packed with cytoplasmic eosinophilic granules. These cells resembled hypertrophied endothelial cells or elongated mast cell analogues. Histochemical staining properties and electron microscopy of these cells are presented. SAV was identified by RT-PCR and neutralizing antibodies against SAV were detected in blood samples. [ABSTRACT FROM AUTHOR]

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses. [ABSTRACT FROM AUTHOR]

Following a natural outbreak of viral encephalopathy and retinopathy (VER) at a commercial farm in Norway, surviving Atlantic halibut, Hippoglossus hippoglossus, were sequentially studied for distribution of nodavirus, immune response and histopathology over 1 year. Typical clinical signs and histopathology of VER were observed during the acute stage of the disease. Most of the surviving fish became subclinical carriers of nodavirus with clusters of nodavirus-containing cells in the central nervous system. Four random samplings of presumably healthy fish were performed from two fish groups, with low and high growth rates respectively, over a 7-month period. Immunohistochemical (IHC) examination revealed a higher number of nodavirus-positive cells in fish with a low growth rate than in fish with a high growth rate. All IHC positive fish were also reverse transcriptase polymerase chain reaction (RT-PCR) positive for nodavirus and for nodavirus antibodies detected by enzyme-linked immunosorbent assay (ELISA) at all sampling points. The percentage of PCR- and ELISA-positive fish remained high throughout the year, while the number of IHC-positive fish decreased, especially in the group with a high growth rate. Several other histopathological changes were observed, including pericarditis, steatitis, changes in liver and kidney, and necrosis of the intestinal wall. None of these findings seemed to be related to the nodavirus infection. Nodavirus was reisolated in cell culture from subclinically infected fish one year after the acute VER outbreak, which indicates that the virus was still infectious. [ABSTRACT FROM AUTHOR]

Infectivity of internal organs and cells of Atlantic salmon, Salmo salar L., was studied at various times after infection with the aetiological agent of infectious salmon anaemia (ISA). Experimentally infected salmon smolts developed anaemia just prior to the onset of ISA-mortality. ISA-infectivity of preparations made from liver, kidney. spleen, plasma red blood cells (RBC) and head kidney leucocytes was determined by inoculating Atlantic salmon part with the respective preparations. ISA-mortality was observed after inoculation of salmon pan with preparations of kidney and liver and to a minor degree, with spleen and a fraction of head kidney leucocytes (WBC1) collected 7 days post-infection. At 11 days post-infection, the infectivity of these preparations increased and ISA-infectivity was also observed with a second fraction of head kidney leucocytes (WBC2), red blood cells (RBC) and blood plasma. At this time, the ISA-infectivity of kidney was not significantly higher (P> 005) than that of liver but was significantly higher than all other preparations as judged by Cox-regression analysis. At days 14 and 18 post-infection, the infectivity of kidney was significantly higher (P < 0.05) than that of liver, but not at 21 and 25 days post-infection. Generally, the ISA-infectivity of kidney was higher than spleen, head kidney leucocytes (WBC1 and WBC2), RBC and plasma, although the difference was not significant at all time points. For example, at day 25 post-infection, the infectivity of kidney was only significantly higher than that of spleen and plasma. On a per gram basis, head kidney leucocytes proved to contain higher amounts of infectious matter than RBC. ISA-infective leucocytes present in the kidney tissue may have contributed to a major part of the infectivity recognized in the kidney preparations. Thus, head kidney leucocytes and other kidney leucocytes also may be considered to be among the most important target cells of the aetiological agent of ISA. [ABSTRACT FROM AUTHOR]

The article presents information on a study analyzing the etiological agent of infectious salmon anemia (ISA) affecting farmed Atlantic salmon in Norway. The most typical signs in the terminal stage of the disease are anemia, leucopenia, ascites and haemorrhagic liver necrosis. According to the research, the aetiological agent is most probably a virus. The target cells of the tentative ISA-virus have not been identified. All attempts to propagate the aetiological ISA-agent in cell cultures have failed.

The proliferative response to mitogens of head kidney leucocytes from Atlantic salmon, Salmo salar L.. experimentally infected with infectious salmon anaemia (ISA) was examined. The mean haematocrits of ISA-inoculated fish were significantly lower than the mean haematocrits of control-inoculated fish at day 14. Mortality in ISA-inoculated fish appeared at day 16 after inoculation. Seven days after inoculation, leucocytes from ISA-inoculated fish showed an increased response to phytohaemagglutinin (PHA) compared to control-inoculated fish, while no change in the response to lipopolysaccharide from Escherichia coli (LPS) could be observed. At days 14 and 22 after inoculation, the responses to both LPS and PHA of lecucocytes from ISA-inoculated fish were severely impaired. These suppressions of the immune response of leucocytes from ISA-inoculatcd fish were found in fish with low haematocrits (<15) as well as in fish with haematocrits higher than 30, suggesting that suppression of the immune system and the development of anaemia are independent events in the pathogenesis of ISA. [ABSTRACT FROM AUTHOR]

In Norway there is an ongoing outbreak in pigs of infections with pandemic influenza A(H1N1)v virus. The first herd was confirmed positive on 10 October 2009. As of 26 October, a total of 23 herds have been diagnosed as positive. The majority of the herds seem to have been infected by humans. Sequence analysis of pig viruses from the index farm shows that they are identical or virtually identical to human viruses from the same geographical region.

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.

Spotted wolffish Anarhichas minor (approx. 0.7 g) were found to be susceptible to infection with a nodavirus isolated from Atlantic halibut (AHNV) by bath-challenge. During the acute stage of infection, 4 to 8 wk post-challenge, viral encephalopathy and retinopathy (VER) were diagnosed by histopathology, immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR). Accumulated mortality was 52% in the challenged group. The surviving fish were sampled 16 wk post-challenge, by which time they had grown to approximately 17 g. No clinical signs of VER were observed in these fish. RT-PCR examination revealed the presence of nodavirus in several organs of the survivors, but no immunopositive cells were detected by IHC. Nodavirus was reisolated from fish at the last sampling in SSN-1 cells, showing that nodavirus retains virulence in persistently infected wolffish for at least 16 wk post-bath-challenge.

Atlantic halibut Hippoglossus hippoglossus, age 8 mo and weighing 20 g, were challenged by either intraperitoneal injection (i.p.) or by bath exposure using nodavirus isolated from Atlantic halibut. Fish were sampled at intervals over a 41 d period, starting on Day 5 post-challenge. Although no clinical disease or mortality was recorded, the data show that nodavirus did successfully propagate in i.p.-challenged fish. Using conventional end-point reverse transcription (RT)-PCR, nodavirus was detected in the kidney of all examined i.p.-challenged fish, and further in the head, heart, liver and posterior intestine of most of these individuals. Quantitative real-time RT-PCR revealed that the amount of virus in head samples from the i.p.-challenged group increased during the experiment. The presence of nodavirus in nervous tissue of i.p.-challenged fish was detected by immunohistochemistry from Day 13 post-challenge. In the retina, virus positive cells were found adjacent to the circumferential germinal zone at the ciliary margin towards the iris. In the brain, a few positive cells were detected in the tectum opticum. An ELISA was developed to detect anti-nodavirus activity in plasma. The method included an optimized coating procedure, which allowed the use of non-purified nodavirus as the coating antigen in a simple indirect ELISA. An anti-nodavirus antibody response was detected from Day 19 post-challenge in i.p.-challenged fish, while a response was not detected in the bath-challenged or control fish. This experiment demonstrates a subclinical nodavirus infection in Atlantic halibut at a post-juvenile stage induced by i.p. injection of virus.

Isolation in cell culture of nodavirus from Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) is described. The cell line SSN-1 was inoculated with tissue material from affected juveniles (60 d after first feeding). Extensive cytopathic effects (CPE) developed approximately 5 d after inoculation, and were also observed after several passages in the same cell line. Cells from infected cultures showed reactivity with an antiserum against sea bass Dicentrarchus labrax nodavirus in an indirect immunofluorescence test. Analysis of infected cells with reverse transcriptase-polymerase chain reaction (RT-PCR) resulted in a product of the predicted size using primers specific for striped jack Pseudocaranx dentex nodavirus. Electron micrographs of infected SSN-1 cells demonstrated virus particles that were approximately less than 30 nm. Challenge of Atlantic halibut larvae (4 d post-hatching) with supernatants from infected SSN-1 cells resulted in development of VER as verified by immunohistochemistry performed on larvae sampled from Day 9 after challenge. The present results show that a nodavirus from Atlantic halibut has been isolated using the SSN-1 cell line and that virus propagated in cell culture retained virulence.

We have investigated the initial steps in the interaction between infectious salmon anemia virus (ISAV) and cultured cells from Atlantic salmon (SHK-1 cell line). Using radioactively or fluorescently labelled viral particles we have studied the binding and fusion kinetics and the effect of pH on binding, uptake, and fusion of ISAV to SHK-1 cells and liposomes. As pH in the medium was reduced from 7.5 to 4.5, the association of virus to the cells was nearly doubled. The same effect of pH was observed when fusion between ISAV and liposomes was analyzed. In addition, the binding of ISAV to intact SHK-1 cells and to cell membrane proteins blotted onto filters was neuraminidase sensitive. However, the increased binding induced by low pH was not neuraminidase sensitive, probably reflecting activation of a fusion peptide at low pH. By using confocal fluorescence microscopy, the increased fusion of fluorescently labelled ISAV with the plasma membrane due to low pH could be demonstrated. When vacuolar pH in the cells was raised during inoculation with chloroquine or ammonium chloride, both electron and confocal microscopy showed accumulation of ISAV in endosomes and lysosomes. Production of infectious virus could be increased by lowering the extracellular pH during infection. Furthermore, chloroquine present during virus inoculation also caused a reduction in the synthesis of viral proteins in ISAV-infected cells as well as in the production of infective virus. These results indicate that ISAV binds to sialic acid residues on the cell surface and that the fusion between virus and cell membrane takes place in the acid environment of endosomes. This provides further evidence for a high degree of similarity between ISAV and influenza virus and extends the basis for the classification of this virus as a member of the Orthomyxoviridae family.

Haemorrhagic kidney syndrome (HKS), a serious disease affecting Atlantic salmon on the east coast of Canada, was determined to be caused by infectious salmon anaemia virus (ISAV) through the isolation of the pathogen on the SHK-1 (salmon head kidney) cell line and confirmation by ISAV-specific immunofluorescent antibody test (IFAT) and reverse transcriptase polymerase chain reaction (RT-PCR). In addition, the defining histopathology of HKS could be reproduced following the injection of material that rendered challenged fish ISAV-positive by cell culture in the absence of any other detectable pathogen. Preliminary nucleotide sequence comparison does not suggest any direct epidemiological connection between the Canadian and Norwegian isolates.

The preparation of the first monoclonal antibody (MAb) against the orthomyxovirus-like infectious salmon anaemia (ISA) virus is described. Characterization of the MAb included isotyping, enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining of virus infected cell cultures (SHK-1 cells), immunoelectron microscopy (IEM) of negatively stained virus preparations, virus neutralization assay and haemagglutination inhibition assay. The MAb reacted with ISA virus preparations both with immunofluorescent staining and in ELISA. No reactions were observed in cell cultures infected with other viruses infecting salmonids including infectious pancreatic necrosis (IPN) virus, viral haemorrhagic septicaemia (VHS) virus and infectious haematopoietic necrosis (IHN) virus. The MAb was also shown to neutralize ISA virus infection in cell cultures and to inhibit the haemagglutination reaction. IEM demonstrated binding to the surface of negatively stained ISA virions. Thus, it is concluded that the MAb binds to the haemagglutinin on the virion surface. Furthermore, using immunofluorescent staining of virus infected cell cultures, reactivity against all the 13 ISA virus strains currently available was demonstrated. Using the MAb, a simple, rapid direct immunofluorescent assay for ISA virus detection and titration in 96-well tissue culture plates was developed. Infectivity titrations by this method correlated well with titration by cytopathic effects. The reliability of the assay was demonstrated by close agreement in virus infectivity titres among different assays for the same virus that were performed on the same day and on different days. A method for detection of viral antigen in cryosections from ISA diseased fish is also reported that may prove useful for the diagnosis and control of ISA.

Atlantic salmon post-smolts were given an intraperitoneal (ip) injection of tissue homogenate of Atlantic salmon fry from an outbreak of infectious pancreatic necrosis (IPN), and cohabitants were given an ip injection of Earle's balanced salt solution (EBSS). Parallel treatment groups were exposed to recurrent episodes of environmental stress by water drainage twice a week. Fish injected with EBSS and non-injected fish were exposed to water drainage. The control fish were left untreated. Mortality due to IPN started 3 weeks after challenge in non-injected and EBSS-injected fish that had been exposed to water drainage. This showed that the fish used in the experiment were covertly infected with IPN virus (IPNV) prior to challenge, although no virus was detected in the fish sampled before the experiment. In fish that received an injection of IPNV, mortality started 5-6 days after challenge, regardless of the presence or absence of stress exposure. The EBSS-injected cohabitants started to die after an additional 5-6 days, also regardless of the presence or absence of stress exposure. The final cumulative mortality in the IPNV-injected fish was significantly lower than in the EBSS-injected cohabitants, thus suggesting that the secondary immune response after injection of IPNV provided more protection than the response after a water-borne infection. No disease outbreak was observed in the control fish.

Infectious salmon anemia (ISA) virus is the cause of infectious salmon anemia in farmed Atlantic salmon. The virus has been shown to contain RNA with structural characteristics similar to those of accepted members of the Orthomyxoviridae. Further biochemical, physiochemical, and morphological characterization of ISA virus was undertaken to clarify its taxonomic position. The virus was found to be sensitive to chloroform, heat, and low pH and agglutinated erythrocytes from fish. Erythrocytes from mammals or birds were not agglutinated. Receptor-destroying enzyme activity was detected, and the nature of this enzyme was suggested to be an acetylesterase. The buoyant density of the virus was 1.18 g/ml in sucrose and CsCl gradients. The maximum rate of virus replication was observed at 15 degrees C, while no virus was produced at 25 degrees C. Actinomycin D inhibited viral replication, and viral antigen was detected in nuclei by immunofluorescence. The addition of trypsin to the culture medium during virus replication had a beneficial effect on virus replication. ISA virus contains four major polypeptides with estimated molecular sizes of 71, 53, 43, and 24 kDa. Electron microscopy revealed structures closely resembling the nucleocapsids of influenza virus. Mushroom-shaped surface projections were a distinctive morphological feature, which differed from the rod-shaped hemagglutinin projections of the influenza viruses. The data reported here support the relationship of ISA virus to the Orthomyxoviridae, although ISA virus differs from influenza viruses in some morphological characteristics and in showing restricted hemagglutination, in different specificity of the receptor-destroying enzyme, in different polypeptide profile, in being unable to replicate at temperatures above 25 degrees C, and in host range.

The genome of infectious salmon anemia virus (ISAV), which infects farmed Atlantic salmon (Salmo salar L.), is characterized here. The virus has an RNA genome, as shown by using specific DNA virus metabolic inhibitors and radioactive in vivo labeling of ISAV nucleic acid. Electrophoresis of [14C]uridine-labeled ISAV RNA revealed that the ISAV genome is segmented. The genome consists of eight segments that range from 1.0 to 2.3 kb, with a total molecular size of approximately 14.5 kb. One ISAV-specific molecular clone, corresponding to the smallest genome segment, was obtained by cDNA cloning of mRNA from an ISAV-infected cell culture. This clone gave a positive hybridization signal on Northern blots of pelleted ISAV. Pretreatment of the ISAV pellet with RNase A resulted in the disappearance of the positive hybridization signal, demonstrating that the genome is single stranded. Reverse transcriptase PCR with primers corresponding to sequences from the molecular clone and target RNA from ISAV-infected and noninfected fish tissues gave specific positive reactions. Alignments of the nucleotide sequence of the molecular clone did not reveal significant homology with any other available sequence in databases. However, the data presented here, together with morphological and replicational properties previously described, indicate that ISAV has a strong resemblance to members of the Orthomyxoviridae family. This is the first thoroughly characterized orthomyxo-like virus isolated from a teleost.

Infectious salmon anaemia (ISA) is a disease of farmed Atlantic salmon (Salmo salar L.) in Norway that affects both erythrocytic and leucocytic cells. Both cell types are possible target cells for the aetiological ISA agent, which is probably a virus. In the present study the distribution and phenotype of leucocyte populations in the spleen and head kidney of Atlantic salmon that were developing ISA have been examined. Frozen tissues were collected from fish at various times after inoculation with ISA-infective material. Immune and enzyme histochemical techniques were used to characterise the response of leucocyte populations. Acid phosphatase positive macrophages predominantly in the red pulp of the spleen appeared to have engulfed erythrocytes at day 4 after infection. Evidence of degradation products of phagocytosed erythrocytes was present in macrophages in red pulp of the spleen at day 7 after infection, in addition to the usual site of erythrophagocytosis in melanomacrophage accumulations. Signs of erythrophagocytosis were not found in the head or body portions of the kidney. The activation of macrophages in the spleen at day 7 was suggested by decreased reactivity for the enzyme 5' nucleotidase. From day 7, clusters of immunoglobulin positive (Ig +) cells were present in the head kidney, while from day 11, the ellipsoids of the spleen showed reactivity for Ig and complement factor C3. These observations are discussed in relation to early immunoglobulin production and possible immune complex trapping. The present results suggest that the leucocyte populations in Atlantic salmon respond to ISA infection through macrophage activation and the initiation of an immune response.

The present study describes light and electron microscopic changes in the liver of Atlantic salmon during the development of infectious salmon anemia (ISA). Atlantic salmon postsmolts weighing 80-100 g were infected by intraperitoneal injections, and liver samples were collected sequentially between day 0 and day 25 post infection (p.i.), with time intervals of 3-4 days. At each collection time, livers from five infected fish and two control fish were examined. Changes involving the perisinusoidal macrophages were observed by transmission electron microscopy, from day 4 p.i. Large vacuoles, containing a fine-granular material with low electron density, accumulated in the cytoplasm. These changes persisted and became more severe throughout the investigation, leading to a considerable increase in the size of the cells. At day 14 p.i., degenerative features of the sinusoidal endothelium were observed. By day 18 p.i., areas of the liver were devoid of a sinusoidal endothelial lining, bringing hepatocytes in direct contact with blood cells. At this stage, the sinusoids were moderately congested. From day 21 p.i., heavy sinusoidal congestion, peliosis hepatis, and degeneration of the hepatocytes were observed. No virus was observed in any of the inhabitant cell types of the liver. Gross and light microscopic changes were first recorded at day 18 p.i., as was a significant decrease in the hematocrit values. By day 25 p.i., characteristic multifocal, confluent, hemorrhagic necroses were present. Results of the present investigation suggest that the liver lesions observed with ISA are not the result of the development of an anemia alone or caused by direct viral damage to hepatocytes. Hepatocellular degeneration succeeded changes in the perisinusoidal macrophages and degeneration of the sinusoidal endothelium. These changes may have impeded the sinusoidal blood flow and hence caused an ischemic hepatocellular necrosis.

A long-term cell line (SHK-1) supporting replication of the causal virus of infectious salmon anaemia (ISA) has been established. The cell line was developed from a culture of Atlantic salmon (Salmo salar L.) head kidney cells. CPE was observed in SHK-1 cells 12-14 days after inoculation with ISA-infective tissue material. The time for CPE to develop decreased after repeated passages of medium from infected cell cultures to new cultures. Transmission trials demonstrated that Atlantic salmon parr developed ISA after intraperitoneal injection of preparations made from infected cells and growth medium. The ISA infectivity of the cell preparations increased with incubation time of inoculated cells. Cell cultures in a second passage were found to have a higher infectivity than the primary inoculated cultures. Virus particles with a diameter of approximately 100-120 nm, and which contained an external envelope and granules were seen in electron micrographs of thin sections of infected cells. Most of the virus particles were located extracellularly close to the cell surface, and in some cases, a connection between virus and plasma membrane could be observed. This indicates that virus particles were released by budding. Enveloped virus particles of 45-140 nm in diameter were seen in abundance in electron micrographs of a negatively stained purified virus preparation. Large, highly pleomorphic particles up to 700 nm in the longest dimension were occasionally observed in unpurified preparations. The evidence is therefore strong that the virus isolated in SHK-1 cells is the aetiological agent of ISA.

A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has been established. The cell line (SHK-1) was developed from a culture of head kidney leucocytes from Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments, ISA infectivity of cell culture medium could be demonstrated from day 5 after infection of SHK-1 cells with ISA-infective tissue homogenate. ISA infectivity of cell culture medium increased following repeated passages of virus. ISA-infected cell cultures develop cytopathic effects (CPE), making quantitation of virus possible. The development of CPE in ISA virus infected cells was inhibited by ammonium chloride, chloroquine and bafilomycin A, suggesting that infection of SHK-1 cells with ISA virus requires a low-pH step.

Rabbit polyclonal antibodies and mouse monoclonal antibodies (MAb) directed against infectious salmon anaemia virus (ISAV) were produced using a virus prepared in a newly established cell line culture developed from Atlantic salmon head kidney (SHK-1) cells. These antibodies were used to establish an indirect fluorescent antibody test for the detection of viral antigens in cell cultures and tissue cryosections. Specific fluorescence was detected in ISAV-infected cell cultures using rabbit polyclonal and MAb. One selected MAb produced specific staining for ISAV in the tissue sections from all the examined organs of ISAV-infected Atlantic salmon 20 d post infection. Fluorescence was mainly found in the endothelial cells and in single cells scattered throughout the parenchyma of the organs.

The intracellular handing of a mannose-terminated glycoprotein taken up in rainbow trout liver cells by receptor-mediated endocytosis has been studied. The intracellular transport and degradation of ovalbumin (OA) were studied by means of subcellular fractionation in Nycodenz gradients and by differential centrifugation following intravenous injection of the ligand. By using OA labelled with(125)I-tyramine cellobiose ((125)I-TC), the subcellular distribution of labelled degradation products could be studied, since they are trapped intracellularly in the organelle where the degradation takes place. (125)I-TC-OA was shortly after injection (45 min) localized in a homogenous population of endosomes. Labelled degradation products firs appeared in an organelle with the same density distribution as the endosomes. In livers homogenized 2h after injection the degradation products appeared in organelles with increasing size and density. After 24h, the degradation products were recovered in at least two populations of lysosomes with a distribution profile which coincided with that of the lysosomal enzyme β-acetylglucosaminidase.The heterogeneous distribution of the late degradation products seemed not to be due to uptake of ligand in different liver cell types as only the parenchymal liver cells took up labelled OA after intravenous injection of the ligand.

1. Plasma of sea char (Salmo alpinus L.) has the ability to esterify cholesterol in vitro. Heat inactivation of the plasma totally inhibited the esterification. 2. Arrhenius plot of the esterification of [3H]cholesterol was linear in the temperature range 5-35 degrees C. 3. Cholesterol esterification in sea char plasma could not be reversibly inhibited by sulphydryl-blocking agents. Total equilibration of the added [3H]cholesterol and the endogenous cholesterol could therefore not be obtained. 4. Comparison of esterification of endogenous and labelled cholesterol revealed that the isotopic method was valid only in qualitative measurements.

125I-labelled asialofetuin injected intravenously into chars (Salmo alpinus L.) was cleared from the blood with a half-time of approx. 60 min. The injected asialofetuin was found to be accumulating in the liver when different organs were dissected out at various time points after the injection. 125I-labelled fetuin was not concentrated in the liver, and the uptake of 125I-labelled asialofetuin was almost completely inhibited by simultaneously injecting an excess unlabelled asialofetuin, indicating a specific uptake mechanism for the galactose-terminated glycoprotein in char liver. Denatured human serum albumin was not taken up by the liver, but was recovered in the kidneys, suggesting that the liver in char is devoid of macrophages. It therefore seems reasonable to assume that the injected 125I-labelled asialofetuin is taken up by the parenchymal cells of the char liver.

1. The initial rate of cholesterol esterification and the concentration of triacylglycerols in plasma were generally lower in prespawning than in immature chars. No typical pattern of variation of plasma cholesterol could be observed. 2. The observed variations in plasma cholesterol esterification and lipids may be caused by reduced feeding in the prespawning period. 3. The initial rate of cholesterol esterification in plasma was positively correlated with the concentrations of triacylglycerols and unesterified cholesterol in plasma, respectively.

1. CoA-dependent cholesterol esterification, measured as esterification of 3H-cholesterol, was demonstrated in homogenates of liver and intestinal mucosa of the char (Salmo alpinus L.). 2. Plasma concentration of triacylglycerols, unesterified and total cholesterol were significantly reduced to 43, 58 and 72% of the control values, respectively, after 6 weeks fasting. 3. The rate of cholesterol esterification in plasma and liver homogenate was significantly lower in the fasted fish compared to the controls, but the esterification activity in the homogenate of intestinal mucosa increased twofold in the fish fasted for 6 weeks.

Modified serum albumin is cleared from the blood by kidney cells in salmonid fishes. The present study deals with isolation of cells from pronephros which endocytose formaldehyde-treated human serum albumin (fHSA). Radioactively labelled fHSA or dinitrophenyl-conjugated albumin (DNP-HSA) were injected intravenously into rainbow trouts. Pronephros cells, containing the endocytosed protein, were isolated and further separated by centrifugal elutriation and density-gradient centrifugation. Most of the radioactive protein was elutriated together with small cells. After centrifuging the cells through a Percoll density gradient, radioactive protein was located in cells recovered in the upper part of the gradient. In mammals, fHSA and other modified proteins are mainly taken up by sinusoidal endothelial cells in the liver via a "scavenger receptor"0. Our results suggest that a comparable function in salmonids is located in a subpopulation of relatively small cells in kidney tissue, possibly sinusoidal lining cells. The separation techniques used seemed to be suitable for isolation of different populations of pronephros cells.

The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal.

In vitro degradation of 125I-formaldehyde treated human serum albumin (fHSA) in char (Salmo alpinus L.) pronephros cells was studied. The labelled protein was injected intravenously and after various intervals of time pronephros cells were isolated and degradation of internalized protein was measured. No degradation could be observed in cells isolated 30 min after injection. The degradation was very effective in cells isolated at later time points (60-90 min); as much as 65% of the initial cell associated labelled protein was degraded during 90 min incubation at 15 degrees C. The effect of temperature on degradation showed a linear course in the temperature range 0-20 degrees C when plotted in an Arrhenius plot. Monensin and ammonium ions inhibited degradation while colchicine had no effect when pronephros cells were isolated 75 min after the injection.

Isolated hepatocytes from rainbow trout and rat were incubated with 14C-labeled linoleic acid, linolenic acid, dihomogammalinolenic acid or eicosapentaenoic acid. The most striking difference in the desaturase activity was the lower level of delta 5 desaturase in trout than in rat. No delta 4 desaturation of 22:4(n-6) to 22:5(n-6) was observed in either of the two species, while the conversion of 22:5(n-3) to 22:6(n-3) was significant in both groups and highest in rainbow trout. The chain-elongating activity was remarkably similar in the two species, except for the "dead-end" elongation which was distinctly more important in fish.

A mannose-terminated glycoprotein,(125)I-invertase, was taken up and degraded by isolated rainbow trout liver cells at 12°C. The uptake was inhibited by EGTA and no degradation occurred in the presence of ammonium ions. The liver cell suspension was fractionated by differential centrifugation in parenchymal and nonparenchymal cells, respectively. The parenchymal liver cells seemed to be the most active cells in uptake of labelled invertase bothin vitro andin vivo. Only negligible amounts of ligand were recovered in the nonparenchymal cells. Internalization of(125)I-invertase at different temperatures was demonstrated indirectly by releasing surface-bound ligand with EGTA. Ligand was internalized even at 0°C in trout liver cells.In vitro uptake of(125)I-invertase was inhibited by excess unlabelled invertase, by mannan and by N-acetylglucosamine.These data suggest that invertase is endocytosed by a mannose-specific pathway by the parenchymal liver cells of rainbow trout.

Intravenously injected 125I-labeled galactose-terminated glycoproteins were mainly recovered in the liver of the rainbow trout. After injection of [14C]sucrose-labeled asialofetuin, the liver cells were isolated and separated by differential centrifugation. The radioactivity was located in the parenchymal cells. Uptake of asialoglycoproteins in liver cells was inhibited by EGTA, lactose and excess unlabeled ligand. Degradation was inhibited by ammonium chloride, suggesting a lysosomal process. Internalization of 125I-asialoglycoproteins was demonstrated by removing receptor-bound ligand with EGTA at different time points during the incubation. The cellular uptake occurred even at 0 degree C.