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.

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.

Rubella serology: a comparison of four methods for exclusion of non-specific serum inhibitors.

The ability of the pyrogenic silica Aerosil 380R to exclude non-specific serum inhibitors (NSI) of rubella virus haemagglutination was evaluated. The developed procedure was compared with the kaolin, heparin/MnCl2 and dextran sulphate/CaCl2 methods. Aerosil and kaolin were found superior for the elimination of non-specific inhibitors and high density lipoproteins (HDL). The other methods left NSI and HDL in a majority of the sera, occasionally in high titres. Aerosil seemed to be somewhat more efficient than kaolin in NSI and HDL exclusion. The Aerosil method offers the opportunity to detect sera with rubella antibody titres less than 10. Among eight such sera, six were shown to contain rubella antibodies, while two were false positives.