The activity of a reference preparation of rainbow trout circulating interferon (IFN), induced by experimental infection of fish with viral haemorrhagic septicaemia virus, was detected on the basis of its non-specific protective effect for RTG-2 cells, grown in microplates and challenged with infectious pancreatic necrosis virus. The IFN was titrated by spectrophotometric assessment of the absorbancy (wavelength 595 nm) of dried cell monolayers, stained with crystal violet after virus challenge. By definition, the reciprocal of the IFN sample dilution giving a cell layer with 50 % of the dye absorbancy of the uninfected control cell layer represented the IFN titre. This spectrophotometric method of determining IFN titre appeared as sensitive as the plaque assay normally used for this purpose, but was better suited to the processing of large numbers of IFN samples, required by investigations on the pathogenes~s of fish viroses. The method proved effective at detecting IFN in homogenates of whole rainbow trout fry, thus allowing individual screening for IFN of small fish which are the usual targets of systemic viroses.
A non-pathogenic cell culture adapted variant was obtained from a normally pathogenic strain of IPN virus (Sp type) after several passages in RTG-2 cells. This cell culture modified (CCA) strain was compared with the original wild (W) strain in various tests. Cross neutralization tests showed no obvious antigenic difference between the two. However the CCA strain was neutralized by a 1:5000 dilution of normal trout serum whereas the W strain was not. In RTG-2 cells, CCA strain gave both large and small plaques, whereas the W strain gave only small ones. Both virus strains were heat sensitive and labile to cyclic freezing and thawing. The CCA virus was more stable in storage at 4°C under different pH conditions and its growth in RTG-2 cells was more rapid. Thert (supraoptimal temperature at which viral yield is depressed by 90%) appeared to be 19-20°C for the CCA strain and 18-19°C for the W strain. Pre-treatment of RTG-2 cells with ultraviolet inactivated CCA virus could inhibit growth of W virus, but had no effect on replication of homologous virus.
In early 1980, perch, Perca fiuviatilis L., were obtained for experimental studies from a pond situated in Sologne, France. One hundred fish (mean weight 19-5 g) were kept in quarantine in a concrete tank (4-8 x 1 m, 0-3 m deep) supplied with tap water dechlorinated through charcoal filters (temperature 10-12°C). Within 1 week of their arrival perch started to display nervous signs including loss of equilibrium and disorganized swimming, followed by death of more than 30% of the total stock. Parasitological and bacteriological examinations gave negative results and a virus isolation was attempted.The anterior kidneys and the spleens of three freshly dead perch were pooled, mixed with sterile sand and ground with mortar and pestle, and diluted 1/20 in Earle's balanced salt solution supplemented with antibiotics following routine procedures. Brains were pooled separately and submitted to the same processing. After centrifugation (5000 §-, 30 min) OT ml of each supernatant was inoculated into 24-h-old RTG-2 (Wolf & Quimby 1962) monolayers (3 x 10^ cells/16 m well of a 24-well Costar plate). Each well received 1 ml of Eagle's minimal essential medium (Stocker's modification) pH 7-6 supplemented with 2% foetal calf serum. Plates were incubated at 14°C and examined daily for cytopathic effect (CPE). No CPE was observed until the ninth day following inoculation when small foci of rounding cells were noticed in wells inoculated with brain material. These foci progressively turned into necrotic plaques similar to the plaques obtained with trout rhabdoviruses (Ghittino & de Kinkelin 1975). Cytopathic efiect gradually spread and destruction of monolayers occurred within 20 days. OT-ml samples from presumably infected wells were then inoculated on to fresh cell monolayers and a similar CPE developed again. Harvest from infected cells was subsequently titrated under agarose medium following the procedure used for IPN or VHS viruses (de Kinkelin & Scherrer 1970). Three days after plaquing, cells were stained with neutral red and small plaques (< 0-5 mm, whereas IPN or VHS plaques ranged from 1 to 2 mm) could be counted. The titres obtained did not exceed 10^ plaque forming units (pfu)/ml. Mass production was attempted in Falcon vials but not achieved, probably due to a too rapid decrease of pH. Production of the putative virus was therefore continued in 24-well plates. It was observed that after more than 20 passages the CPE appeared progressively earlier (finally 4 days after inoculation). 241 242 M. Dorson et al.appeared, but sometimes small plaques of rounding cells were observed which subsequently healed. A 10^ pfu/ml virus suspension heated 30 min at 37°C underwent a titre decline of at least 99%.Electron microscopy was carried out on RTG-2 cells 4 days post-infection when CPE was just appearing. Cells were fixed in 1-6% glutaraldehyde buffered with 0-1 M sodium cacodylate (pH 7-3), post-fixed in 1 % osmium tetroxide and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed on a Ph...
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