Persistent infections with infectious pancreatic necrosis virus (IPNV) of different virulence in Atlantic salmon, Salmo salar L.

Infectious pancreatic necrosis virus (IPNV) is a prevalent pathogen in fish worldwide. The virus causes substantial mortality in Atlantic salmon juveniles and smolts when transferred to sea water and persistent infection in surviving fish after disease outbreaks. Here, we have investigated the occurrence of the virus as well as the innate immune marker Mx in the head kidney (HK) of Atlantic salmon throughout an experimental challenge covering both a fresh and a seawater phase. The fish were challenged with a high (HV) and low virulence (LV) IPNV. Both isolates caused mortality due to reactivation of the virus after transfer to sea water. In the freshwater phase, higher levels of virus transcripts were detected in the HK of fish infected with LV IPNV compared to HV, suggesting that the HV isolate is able to limit its own replication to a level where the innate immune system is not alerted. Further, ex vivoHK leucocytes derived from fish infected with the two isolates were stimulated with CpG DNA. Significantly, higher IFN levels were found in the LV compared to the HV group in the freshwater phase. This suggests that the viruses attenuate the antiviral host immune response at different levels which may contribute to the observed differences in disease outcome.

Establishing a cell line from Atlantic cod as a novel tool for in vitro studies.

The present work describes the generation of a cell line from newly hatched Atlantic cod (Gadus morhua) larvae (ACL cells). Primary cultures were initiated by explant outgrowth from partially minced tissues and subcultured cells were exposed to UV radiation. After a substantial period of growth lag, cells started to proliferate and different growth conditions were tested to establish the cell line. At present, the ACL cell line has been subcultured for more than 100 passages. ACL cells had a polygonal shape and the morphology appeared homogenous with epithelial-like cells. Cell growth was dependent on the presence of foetal bovine serum and cells proliferated in a wide temperature range with optimal growth at 15 °C. By exposure to a viral dsRNA mimic (poly I:C) the cells expressed high levels of a repertoire of genes comprising both inflammatory mediators and interferon stimulated genes. Infection studies with two different viruses showed that infectious pancreatic necrosis virus (IPNV) propagated efficiently, and induced low level expression of genes of both pathways before the cells rapidly died. No productive infection was obtained with nervous necrosis virus (NNV), but a transient increase in the viral RNA level, followed by a high increase in expression of selected ISGs, suggests that the virus enters the cells but is unable to complete its replication cycle. To our knowledge, ACL cells are at the moment the only existing cell line from Atlantic cod. Our results demonstrate that ACL cells can be a useful research tool for further exploration of host-pathogen interactions and it is believed that this cell line will serve as a valuable tool also for studies within other research areas.

Study of virulence in field isolates of infectious pancreatic necrosis virus obtained from the northern part of Norway.

In order to study the variety of infectious pancreatic necrosis virus (IPNV) strains involved in outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon fish farms, samples were collected from 19 different outbreaks of IPN in the northern part of Norway. The main objective of this study was to examine whether IPNV isolates of different virulence were involved in the outbreaks and could explain the variable IPN protection observed in vaccinated post-smolts in the field. Both the molecular basis of virulence of all field isolates and virulence expressed by mortality after bath challenge of unvaccinated post-smolts with eight of the isolates were studied. Very little variation among the field isolates was detected when the 578-bp variable region encoding the VP2 protein known to be involved in virulence was sequenced. The cumulative mortality after experimental challenge with field isolates genetically characterized as highly virulent was always high (40-56%), while the cumulative mortality of the same strains in vaccinated post-smolts during the field outbreaks varied from 1 to 50%. Although the tested samples came from fish vaccinated with the same vaccine product, the protection against IPN varied. These results demonstrate that differences in virulence of the isolates were not the main reason for the variation in mortality in the field outbreaks. Most of the field isolates were of high virulence, which is shown in experimental challenges to be important for mortality, but clearly other factors that might affect the susceptibility of IPN also play an important role in the outcome of an IPNV infection.

Reference genes evaluated for use in infectious pancreatic necrosis virus real-time RT-qPCR assay applied during different stages of an infection.

The stability of 6 reference genes, 18S, beta-actin, RPS20, eEF1alpha, G6PDH and GAPDH, was examined in tissues from Atlantic salmon (Salmo salar) and Chinook salmon embryo cells (CHSE-214). The main objective of this study was to determine the most suitable reference genes for use for the normalisation of data in quantitative real-time RT-qPCR assays conducted on infected tissues. The tissue samples selected for analysis were taken from head kidney and pylorus and collected at different time points during a challenge experiment with infectious pancreatic necrosis virus (IPNV). The stability of some of the reference genes was also studied in infected CHSE-214 cells. The ranking of the genes examined was carried out using the geNorm program. This program determines the most stable genes from a set of genes tested in a given cDNA sample. The stability of the reference genes varied in different tissues and in the cell line at different stages of infection with IPNV. This study demonstrated that tissue-specific combinations of reference genes must be used to normalise real time data for use for the quantitation of IPNV.

Studies on uptake and intracellular processing of infectious pancreatic necrosis virus by Atlantic cod scavenger endothelial cells.

Previous work in our group has identified the scavenger endothelial cells (SECs) of heart endocardium in cod, Gadus morhua L., as the major site for elimination of both physiological and foreign macromolecular waste from the circulation. The present study was undertaken to establish the role of cod SECs in the clearance of virus. We focused on infectious pancreatic necrosis virus (IPNV) as it is a well-known virus with a broad host range, and causes significant economic losses in the salmon industry. Our results showed that cod SEC cultures infected by the IPNV produce high titres of new virus. Ligand-receptor inhibition experiments suggested that the virus did not enter the cells through any of the major endocytosis receptors of cod SECs. Yet, the infection lowered the capacity of the cells to endocytose ligands via the scavenger receptor. Inhibitors of receptor recycling and vesicle acidification did not affect virus infectivity. The finding that SEC cultures prepared from 25% of the cod produced high titres of IPNV without being infected in the laboratory, suggests that SECs of cod may serve as reservoirs for IPNV in persistently infected cod.

Acute and persistent experimental nodavirus infection in spotted wolffish Anarhichas minor.

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.

CpG oligodeoxynucleotides and plasmid DNA stimulate Atlantic salmon (Salmo salar L.) leucocytes to produce supernatants with antiviral activity.

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report herein that plasmid DNA and synthetic oliogodeoxynucleotides (ODNs) containing unmethylated CpG induce production of antiviral cytokine activity in Atlantic salmon leucocytes, whereas ODNs with an inverted motif (GpC) or with methylated cytosines have nearly no stimulatory effect. The adherent cell population, representing mainly macrophages, is directly activated by CpG-ODN, while the effect on the non-adherent population is weak. Since the peak antiviral activity in ODN-stimulated leucocytes is seen after 48h, this might indicate that the unmethylated DNA stimulates the adherent cells to produce co-stimulatory molecules, which in turn stimulates production of antiviral cytokines in the non-adherent cell population. The potent immune activation by CpG ODNs points to possible new applications as adjuvant in fish vaccines.

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.

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells.

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.

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

Infectious salmon anaemia virus (ISAV), which previously had never been isolated in any of the commercially available established fish cell lines, was successfully propagated in the continuous cell line Atlantic salmon (AS). The yield of infectious ISAV increased with the incubation time of virus-inoculated cells, demonstrated by in vivo infectivity trials in groups of Atlantic salmon. Trypsin treatment of the virus was not necessary for primary infection of AS cells with salmon-grown ISAV. The infection was non-cytopathic, but it was possible to detect virus-infected cells by a haemadsorption centre assay using Atlantic salmon erythrocytes. Pleomorphic enveloped virus particles were seen by transmission electron microscopy of infected AS cells. Elongated forms were observed, but spherical particles with diameters of 90-130 nm were commonest. Growth of ISAV was inhibited by actinomycin D but not by 5-bromo-2-deoxyuridine treatment, which indicates that ISAV may be an aquatic orthomyxovirus.

Is Helicobacter pylori the cause of dyspepsia?

OBJECTIVE: To determine the association between infection with Helicobacter pylori and dyspepsia. DESIGN: Cross sectional study of dyspeptic subjects and age and sex matched controls identified by a questionnaire survey of all inhabitants aged 20-69. (Endoscopy, histological examination, and microbiological examinations of biopsies from the gastric mucosa were performed blind.) SETTING: Population based survey in Sørreisa, Norway. SUBJECTS: All 782 dyspeptic subjects (excluding those with a previous history of peptic ulcer, gall stones or kidney stones, and coronary heart disease) and controls were offered an endoscopy, of whom 309 dyspeptic subjects and 310 controls attended. MAIN OUTCOME MEASURES: Prevalences of endoscopic and histological diagnoses and of cultures positive for H pylori. RESULTS: A high prevalence of positive cultures, increasing with age, was found in both dyspeptic subjects (48%) and non-dyspeptic controls (36%) (p = 0.004). Positive cultures in both dyspeptic subjects and controls were strongly associated with histological gastritis (70%, 95% confidence interval 65.5 to 85.3; 60%, 52.7 to 67.7, respectively) and peptic ulcer (92%, 61.5 to 99.8; 64.1, 9.4 to 99.2, respectively). Only 3% of subjects with a histologically non-inflamed gastric mucosa had this infection (dyspeptic subjects 2%, 0.2 to 7.0; controls 4%; 1.2 to 8.8). CONCLUSIONS: The relation between dyspeptic symptoms and H pylori is dubious; H pylori seems to have a pathogenetic role in gastritis and may be a contributing factor but not a cause of peptic ulcer.

Hantaviral antigens and antibodies in wild rodents in Portugal.

Small rodents of the species Rattus norvegicus and Rattus rattus have been captured between 1986 and 1988 in several areas of Southern Portugal. A total of 135 animal specimens were examined for hantaviral antigens in lung sections and 5 have been found positive. Some of the rodents were shown to have serum antibodies as detected by immunofluorescence in titres up to 1:256. This investigation proves for the first time the presence of Hantavirus in wild rodent populations of Portugal.

Hemorrhagic fever with renal syndrome (nephropathia epidemica) in Norway: seroepidemiology 1981-1985.

An indirect fluorescent antibody technique (IFAT) was applied to serologically confirm the clinical diagnosis in 507 nephropathia epidemica (NE) suspected patients. Hantaan virus (HV), the agent of Korean hemorrhagic fever, which is serologically related to the NE agent, was used as antigen. Both IgG and IgM reactions were detected. High levels of IgG antibodies to HV were common, even in the acute phase of illness. Over a 5-year period, a total of 35% of the NE suspected patients revealed antibodies to HV, but this varied considerably in the different years. In 2 endemic areas the serological confirmation of the NE diagnosis was 60%. 82% of the seropositive NE patients lived in 4 endemic areas. The bank vole (Clethrionomys glareolus) was common in all areas and predominant in 2. The wood mouse (Apodemus sylvaticus) was abundant in the other 2. In years with high bank vole population, the number of seropositive NE cases increased, with a peak in October/November. When the bank vole population was low, the relatively few seropositive NE cases occurred more regularly throughout the year. Subclinical infections were common. Antibodies to HV were detected in 74% male and 26% female NE patients, but the ratio varied between age groups.

Reservoir animals for nephropathia epidemica in Norway: indications of a major role for the bank vole (C. glareolus) in comparison with the woodmouse (A. sylvaticus).

Small rodents were collected live in two different locations within a nephropathia epidemica (NE) endemic area, and tested for both antiviral serum antibodies and viral antigens in lung sections. In one location, only Apodemus sylvaticus (woodmice) were found in the traps, in the other, both A. sylvaticus and Clethrionomys glareolus (bank voles) were collected. Among the woodmice from the former location the prevalence of NE virus markers was significantly lower than for either woodmice or bank voles from the other location, and no NE antigen-positive animals was found. The woodmice co-existing with bank voles had a lower prevalence of NE antigen and antibodies than the bank voles, and fewer woodmice had both antibodies and antigen. The results emphasize the important role of bank voles as a major NE virus reservoir and probable source of human infections.

Nephropathia epidemica in Norway: antigen and antibodies in rodent reservoirs and antibodies in selected human populations.

Nephropathia epidemica (NE) antigen was detected by IFAT (indirect fluorescent antibody technique) in the lungs of 14 of 97 bank voles (Clethrionomys glareolus) collected in three endemic areas. The distribution of antigen positive voles within an endemic location was scattered. Antibodies to Korean hemorrhagic fever (KHF) virus antigens were detected by IFAT in 12 of 14 NE antigen positive bank voles and in 15 of 83 that were antigen negative. NE antigen positive voles exhibited higher antibody titres. Antibodies to KHF were demonstrated in sera from C. rutilus and C. rufocanus collected more than 200 km north of the distribution area for C. glareolus. It appears likely that these vole species can serve as virus vectors for NE cases occurring north of the bank vole area. NE antibodies cross-reacting with KHF virus seem to diminish with time after infection in some NE patients, while for others such cross-reacting antibodies were detected up to 12 years after the disease. Antibodies to KHF were detected in eight of 106 healthy forestry workers with no clinical history of NE. No serological cross-reactions were detected between NE/KHF antigens and representative Bunyaviridae present in Norway. NE/KHF-like viruses appear widespread in Norway, both within and outside of the distribution area of the bank vole.

Lack of cross-reactivity between antibody against smooth muscle and antibodies to measles virus in sera from patients with chronic active hepatitis.

A majority of patients with HBsAg-negative chronic active hepatitis (CAH) show considerably raised titres of serum antibodies against measles virus antigens. Since these patients often have high titers of antiactin specific antibodies against smooth muscle, and since actin is also incorporated into the measles virion during replication, we have examined whether there was any cross-reactivity between the two antigen-antibody systems. Cross-absorptions of sera from patients with CAH were performed using measles virus antigens and smooth muscle from human uterus. The corresponding specific antibodies were removed from serum without significantly affecting the titres of the other antibodies. Absorption had no influence on the total amount of IgG in serum. The raised titres of antibodies against measles virus seen in CAH can therefore not be explained by cross-reaction with antibodies to smooth muscle.